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Sample records for immunoglobulin omega light-chain

  1. Atypical immunoglobulin light chain amyloidosis

    PubMed Central

    Wu, Xia; Feng, Jun; Cao, Xinxin; Zhang, Lu; Zhou, Daobin; Li, Jian

    2016-01-01

    Abstract Background: Primary immunoglobulin light chain amyloidosis (AL amyloidosis) is a plasma cell disorder which mainly affects heart, kidneys, liver, and peripheral nervous system. Cases of atypical AL amyloidosis presented as spontaneous vertebral compression fractures have been rarely reported, and data about the management and clinical outcomes of the patients are scarce. Methods: Herein, we present 3 new cases of AL amyloidosis with spontaneous vertebral compression fracture and review 13 cases retrieved from the literature. Results: Moreover, we observed overrepresentations of liver involvement and bone marrow involvement in AL amyloidosis with spontaneous vertebral compression fracture. Conclusion: We believe that better awareness of the rare clinical presentation as spontaneous vertebral compression fracture of AL amyloidosis can facilitate earlier diagnosis and earlier treatment. PMID:27603350

  2. Immunoglobulin light chains, glycosaminoglycans and amyloid.

    SciTech Connect

    Stevens, F. J.; Kisilevsky, R.; Biosciences Division; Queen's Univ.

    2000-03-01

    Immunoglobulin light chains are the precursor proteins for fibrils that are formed during primary amyloidosis and in amyloidosis associated with multiple myeloma. As found for the approximately 20 currently described forms of focal, localized, or systemic amyloidoses, light chain-related fibrils extracted from physiological deposits are invariably associated with glycosaminoglycans, predominantly heparan sulfate. Other amyloid-related proteins are either structurally normal, such as g2-microglobulin and islet amyloid polypeptide, fragments of normal proteins such as serum amyloid A protein or the precursor protein of the g peptide involved in Alzheimer's disease, or are inherited forms of single amino acid variants of a normal protein such as found in the familial forms of amyloid associated with transthyretin. In contrast, the primary structures of light chains involved in fibril formation exhibit extensive mutational diversity rendering some proteins highly amyloidogenic and others non-pathological. The interactions between light chains and glycosaminoglycans are also affected by amino acid variation and may influence the clinical course of disease by enhancing fibril stability and contributing to resistance to protease degradation. Relatively little is currently known about the mechanisms by which glycosaminoglycans interact with light chains and light-chain fibrils. It is probable that future studies of this uniquely diverse family of proteins will continue o shed light on the processes of amyloidosis, and contribute as well to a greater understanding of the normal physiological roles of glycosaminoglycans.

  3. Interaction between glycosaminoglycans and immunoglobulin light chains.

    SciTech Connect

    Jiang, X.; Myatt, E.; Lykos, P.; Stevens, F. J.; Center for Mechanistic Biology and Biotechnology; Illinois Inst. of Tech.

    1997-01-01

    Amyloidosis is a pathological process in which normally soluble proteins polymerize to form insoluble fibrils (amyloid). Amyloid formation is found in a number of diseases, including Alzheimer's disease, adult-onset diabetes, and light-chain-associated amyloidosis. No pharmaceutical methods currently exist to prevent this process or to remove the fibrils from tissue. The search for treatment and prevention methods is hampered by a limited understanding of the biophysical basis of amyloid formation. Glycosaminoglycans (GAGs) are long, unbranched heteropolysaccharides composed of repeating disaccharide subunits and are known to associate with amyloid fibrils. The interaction of amyloid-associated free light chains with GAGs was tested by both size-exclusion high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments. The results indicated that heparin 16 000 and chondroitin sulfate B and C precipitated both human intact light chains and recombinant light chain variable domains. Although all light chains interacted with heparin, the strongest interactions were obtained with proteins that had formed amyloid. Molecular modeling indicated the possibility of interaction between heparin and the conserved saddle like surface of the light chain dimer opposite the complementarity-determining segments that form part of the antigen-binding site of a functional antibody. This suggestion might offer a new path to block the aggregation of amyloid-associated light chain proteins, by design of antagonists based on properties of GAG binding. A hexasaccharide was modeled as the basis for a possible antagonist.

  4. Comparison of serum free light chain and urine electrophoresis for the detection of the light chain component of monoclonal immunoglobulins in light chain and intact immunoglobulin multiple myeloma.

    PubMed

    Dejoie, Thomas; Attal, Michel; Moreau, Philippe; Harousseau, Jean-Luc; Avet-Loiseau, Herve

    2016-03-01

    Response criteria for multiple myeloma are based upon changes in monoclonal protein levels quantified using serum and/or urine protein electrophoresis. The latter lacks sensitivity at low monoclonal protein levels and since 2001, the serum free light chain test has been available and its clinical utility proven, yet guidelines have not recommended it as a replacement for urine assessment. Herein we evaluated responses using serum free light chain measurements and serum and urine electrophoresis after 2 and 4 cycles of therapy and after stem cell transplantation in 25 light chain and 157 intact immunoglobulin myeloma patients enrolled in the IFM 2007-02 MM trial. All 25 light chain patients had measurable disease by serum free light chain and urine methods at presentation. By contrast 98 out of 157 intact immunoglobulin patients had measurable disease by serum free light chain compared to 55 out of 157 by urine electrophoresis. In all patients there was substantial agreement between predicate (serum/urine protein electrophoresis) and test (serum protein electrophoresis and serum free light chain) methods for response assessment (Weighted Kappa=0.83). Urine immunofixation became negative in 47% light chain and 43% intact immunoglobulin patients after 2 cycles of therapy. At this time the serum free light chain ratio normalised in only 11% and 27% patients, respectively. In summary we found good agreement between methods for response assessment, but the serum free light chain test provided greater sensitivity than urine electrophoresis for monitoring. To our knowledge this is the first report comparing both methods for response assignment based on the International Myeloma Working Group guidelines. (Clinical Trials Register.eu identifier: 2007-005204-40).

  5. Shared epitopes of avian immunoglobulin light chains.

    PubMed

    Benčina, Mateja; Cizelj, Ivanka; Berčič, Rebeka Lucijana; Narat, Mojca; Benčina, Dušan; Dovč, Peter

    2014-04-15

    Like all jawed vertebrates, birds (Aves) also produce antibodies i.e. immunoglobulins (Igs) as a defence mechanism against pathogens. Their Igs are composed of two identical heavy (H) and light (L) chains which are of lambda isotype. The L chain consists of variable (VL), joining (JL) and constant (CL) region. Using enzyme immunoassays (EIA) and two monoclonal antibodies (mAbs) (3C10 and CH31) to chicken L chain, we analysed their cross-reactivity with sera from 33 avian species belonging to nine different orders. Among Galliformes tested, mAbs 3C10 and CH31 reacted with L chains of chicken, turkey, four genera of pheasants, tragopan and peafowl, but not with sera of grey partridge, quail and Japanese quail. Immunoglobulins of guinea-fowl reacted only with mAb 3C10. Both mAbs reacted also with the L chain of Eurasian griffon (order Falconiformes) and domestic sparrow (order Passeriformes). Sera from six other orders of Aves did not react with either of the two mAbs. EIA using mAbs 3C10 and CH31 enabled detection of antibodies to major avian pathogens in sera of chickens, turkeys, pheasants, peafowl, Eurasian griffon and guinea-fowl (only with mAb 3C10). The N-terminal amino acid sequence of pheasant L chain (19 residues) was identical to that of chicken. Sequences of genes encoding the L chain constant regions of pheasants, turkey and partridge were determined and deposited in the public database (GenBank accession numbers: FJ 649651, FJ 649652 and FJ 649653, respectively). Among them, amino acid sequence of pheasants is the most similar to that of chicken (97% similarity), whereas those of turkey and partridge have greater similarity to each other (89%) than to any other avian L chain sequence. The characteristic deletion of two amino acids which is present in the L chain constant region in Galliformes has been most likely introduced to their L chain after their divergence from Anseriformes.

  6. Shared epitopes of avian immunoglobulin light chains.

    PubMed

    Benčina, Mateja; Cizelj, Ivanka; Berčič, Rebeka Lucijana; Narat, Mojca; Benčina, Dušan; Dovč, Peter

    2014-04-15

    Like all jawed vertebrates, birds (Aves) also produce antibodies i.e. immunoglobulins (Igs) as a defence mechanism against pathogens. Their Igs are composed of two identical heavy (H) and light (L) chains which are of lambda isotype. The L chain consists of variable (VL), joining (JL) and constant (CL) region. Using enzyme immunoassays (EIA) and two monoclonal antibodies (mAbs) (3C10 and CH31) to chicken L chain, we analysed their cross-reactivity with sera from 33 avian species belonging to nine different orders. Among Galliformes tested, mAbs 3C10 and CH31 reacted with L chains of chicken, turkey, four genera of pheasants, tragopan and peafowl, but not with sera of grey partridge, quail and Japanese quail. Immunoglobulins of guinea-fowl reacted only with mAb 3C10. Both mAbs reacted also with the L chain of Eurasian griffon (order Falconiformes) and domestic sparrow (order Passeriformes). Sera from six other orders of Aves did not react with either of the two mAbs. EIA using mAbs 3C10 and CH31 enabled detection of antibodies to major avian pathogens in sera of chickens, turkeys, pheasants, peafowl, Eurasian griffon and guinea-fowl (only with mAb 3C10). The N-terminal amino acid sequence of pheasant L chain (19 residues) was identical to that of chicken. Sequences of genes encoding the L chain constant regions of pheasants, turkey and partridge were determined and deposited in the public database (GenBank accession numbers: FJ 649651, FJ 649652 and FJ 649653, respectively). Among them, amino acid sequence of pheasants is the most similar to that of chicken (97% similarity), whereas those of turkey and partridge have greater similarity to each other (89%) than to any other avian L chain sequence. The characteristic deletion of two amino acids which is present in the L chain constant region in Galliformes has been most likely introduced to their L chain after their divergence from Anseriformes. PMID:24603015

  7. Chronic myopathy due to immunoglobulin light chain amyloidosis

    PubMed Central

    Manoli, Irini; Kwan, Justin Y.; Wang, Qian; Rushing, Elisabeth J.; Tsokos, Maria; Arai, Andrew E.; Burch, Warner M.; Dispenzieri, Angela; McPherron, Alexandra C.; Gahl, William A.

    2013-01-01

    Amyloid myopathy associated with a plasma cell dyscrasia is a rare cause of muscle hypertrophy. It can be a challenging diagnosis, since pathological findings are often elusive. In addition, the mechanism by which immunoglobulin light-chain deposition stimulates muscle overgrowth remains poorly understood. We present a 53–year old female with a 10-year history of progressive generalized muscle overgrowth. Congo-red staining and immunohistochemistry revealed perivascular lambda light chain amyloid deposits, apparent only in a second muscle biopsy. The numbers of central nuclei and satellite cells were increased, suggesting enhanced muscle progenitor cell formation. Despite the chronicity of the light chain disease, the patient showed complete resolution of hematologic findings and significant improvement of her muscle symptoms following autologous bone marrow transplantation. This case highlights the importance of early diagnosis and therapy for this treatable cause of a chronic myopathy with muscle hypertrophy. PMID:23465863

  8. Dual immunoglobulin light chain B cells: Trojan horses of autoimmunity?

    PubMed

    Pelanda, Roberta

    2014-04-01

    Receptor editing, a major mechanism of B cell tolerance, can also lead to allelic inclusion at the immunoglobulin light chain loci and the development of B cells that coexpress two different immunoglobulin light chains and, therefore, two antibody specificities. Most allelically included B cells express two κ chains, although rare dual-λ cells are also observed. Moreover, these cells typically coexpress an autoreactive and a nonautoreactive antibody. Thus, allelically included B cells could operate like 'Trojan horses': expression and function of the nonautoreactive antigen receptors might promote their maturation, activation, and terminal differentiation into effector cells that also express and secrete autoantibodies. Indeed, dual-κ B cells are greatly expanded into effector B cell subsets in some autoimmune mice, thus indicating they might play an important role in disease.

  9. Specific dimerization of the light chains of human immunoglobulin.

    PubMed

    Stevenson, G T; Straus, D

    1968-07-01

    1. The light chains of human immunoglobulin were allowed to dimerize in vitro on removal of the dispersing agents acetic acid or urea. 2. On electrophoresis in polyacrylamide gel at pH8.8 the dimers yielded up to nine regularly spaced bands. This approximates to the number of electrophoretic components known to occur among the monomers. 3. Single electrophoretic components of the dimers were isolated from the gel, dissociated into monomers, and subjected as such to electrophoresis in urea-containing gels. Each gave two adjacent bands. 4. Similarly, after all the light chains as monomers had been subjected to electrophoresis in urea-containing gels, single electrophoretic components were isolated and allowed to dimerize. When examined now as dimers in the absence of urea, each component gave two adjacent bands. 5. These findings are explicable on the following basis. (a) The dimerization of the light chains is specific, at least inasmuch as it occurs between monomers of the same electrophoretic mobilities. (b) With the buffer constant, different light chains undergo different changes in net charge on being transferred from urea-containing to urea-free solution; in this way two different chains of the same initial charge can acquire a charge difference of 1. 6. Experiments with Bence-Jones proteins and other homogeneous light chains gave results substantiating the conclusions (a) and (b). PMID:4174431

  10. Increased Serum Free Light Chains Precede the Presentation of Immunoglobulin Light Chain Amyloidosis

    PubMed Central

    Weiss, Brendan M.; Hebreo, Joseph; Cordaro, Daniel V.; Roschewski, Mark J.; Baker, Thomas P.; Abbott, Kevin C.; Olson, Stephen W.

    2014-01-01

    Purpose Patients with immunoglobulin light chain amyloidosis (AL amyloidosis) generally present with advanced organ dysfunction and have a high risk of early death. We sought to characterize monoclonal immunoglobulin (M-Ig) light chains before clinical presentation of AL amyloidosis. Patients and Methods We obtained prediagnostic sera from 20 cases with AL amyloidosis and 20 healthy controls matched for age, sex, race, and age of serum sample from the Department of Defense Serum Repository. Serum protein electrophoresis with immunofixation and serum free light chain (FLC) analysis were performed on all samples. Results An M-Ig was detected in 100% of cases and 0% of controls (P < .001). The M-Ig was present in 100%, 80%, and 42% of cases at less than 4 years, 4 to 11 years, and more than 11 years before diagnosis, respectively. The median FLC differential (FLC-diff) was higher in cases compared with controls at all time periods, less than 4 years (174.8 v 0.3 mg/L; P < .001), 4 to 11 years (65.1 v 2.2 mg/L; P < .001), and more than 11 years (4.5 v 0.4 mg/L; P = .03) before diagnosis. The FLC-diff was greater than 23 mg/L in 85% of cases and 0% of controls (P < .001). The FLC-diff level increased more than 10% per year in 84% of cases compared with 16% of controls (P < .001). Conclusion Increase of FLCs, including within the accepted normal range, precedes the development of AL amyloidosis for many years. PMID:25024082

  11. Targeted disruption of the porcine immunoglobulin kappa light chain locus.

    PubMed

    Ramsoondar, J; Mendicino, M; Phelps, C; Vaught, T; Ball, S; Monahan, J; Chen, S; Dandro, A; Boone, J; Jobst, P; Vance, A; Wertz, N; Polejaeva, I; Butler, J; Dai, Y; Ayares, D; Wells, K

    2011-06-01

    Inactivation of the endogenous pig immunoglobulin (Ig) loci, and replacement with their human counterparts, would produce animals that could alleviate both the supply and specificity issues of therapeutic human polyclonal antibodies (PAbs). Platform genetics are being developed in pigs that have all endogenous Ig loci inactivated and replaced by human counterparts, in order to address this unmet clinical need. This report describes the deletion of the porcine kappa (κ) light chain constant (Cκ) region in pig primary fetal fibroblasts (PPFFs) using gene targeting technology, and the generation of live animals from these cells via somatic cell nuclear transfer (SCNT) cloning. There are only two other targeted loci previously published in swine, and this is the first report of a targeted disruption of an Ig light chain locus in a livestock species. Pigs with one targeted Cκ allele (heterozygous knockout or ±) were bred together to generate Cκ homozygous knockout (-/-) animals. Peripheral blood mononuclear cells (PBMCs) and mesenteric lymph nodes (MLNs) from Cκ -/- pigs were devoid of κ-containing Igs. Furthermore, there was an increase in lambda (λ) light chain expression when compared to that of wild-type littermates (Cκ +/+). Targeted inactivation of the Ig heavy chain locus has also been achieved and work is underway to inactivate the pig lambda light chain locus.

  12. Risk factors for venous thromboembolism in immunoglobulin light chain amyloidosis

    PubMed Central

    Bever, Katherine M.; Masha, Luke I.; Sun, Fangui; Stern, Lauren; Havasi, Andrea; Berk, John L.; Sanchorawala, Vaishali; Seldin, David C.; Sloan, J. Mark

    2016-01-01

    Patients with immunoglobulin light chain amyloidosis are at risk for both thrombotic and bleeding complications. While the hemostatic defects have been extensively studied, less is known about thrombotic complications in this disease. This retrospective study examined the frequency of venous thromboembolism in 929 patients with immunoglobulin light chain amyloidosis presenting to a single referral center, correlated risk of venous thromboembolism with clinical and laboratory factors, and examined complications of anticoagulation in this population. Sixty-five patients (7%) were documented as having at least one venous thromboembolic event. Eighty percent of these patients had events within one year prior to or following diagnosis. Lower serum albumin was associated with increased risk of VTE, with a hazard ratio of 4.30 (CI 1.60–11.55; P=0.0038) for serum albumin less than 3 g/dL compared to serum albumin greater than 4 g/dL. Severe bleeding complications were observed in 5 out of 57 patients with venous thromboembolism undergoing treatment with anticoagulation. Prospective investigation should be undertaken to better risk stratify these patients and to determine the optimal strategies for prophylaxis against and management of venous thromboembolism. PMID:26452981

  13. The immunoglobulin light chain locus of the turkey, Meleagris gallopavo.

    PubMed

    Bao, Yonghua; Wu, Sun; Zang, Yunlong; Wang, Hui; Song, Xiangfeng; Xu, Chunyang; Xie, Bohong; Guo, Yongchen

    2012-06-15

    To date, most jawed vertebrate species encode more than one immunoglobulin light (IgL) chain isotypes. It has been shown that several bird species (chickens, white Pekin or domestic duck, and zebra finches) exclusively express lambda isotype. We analyze here the genomic organization of another bird species turkey IgL genes based on the recently released genome data. The turkey IgL locus located on chromosome 17 spans approximately 75.2kb and contains a single functional V(λ) gene, twenty V(λ) pseudogenes, and a single functional J(λ)-C(λ) block. These data suggest that the genomic organization of bird IgL chain genes seems to be conserved. Ten cDNA clones from turkey Igλ chain containing almost full-length V(λ), J(λ) and C(λ) segments were acquired. The comparison of V(λ) cDNA sequences to all the germline V(λ) segments suggests that turkey species may be generating IgL chain diversity by gene conversion and somatic hypermutation like the chicken. This study provides insights into the immunoglobulin light chain genes in another bird species.

  14. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  15. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  16. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  17. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  18. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  19. Immunoglobulin K light chain deficiency: A rare, but probably underestimated, humoral immune defect.

    PubMed

    Sala, Pierguido; Colatutto, Antonio; Fabbro, Dora; Mariuzzi, Laura; Marzinotto, Stefania; Toffoletto, Barbara; Perosa, Anna R; Damante, Giuseppe

    2016-04-01

    Human immunoglobulin molecules are generated by a pair of identical heavy chains, which identify the immunoglobulin class, and a pair of identical light chains, Kappa or Lambda alternatively, which characterize the immunoglobulin type. In normal conditions, Kappa light chains represent approximately 2/3 of the light chains of total immunoglobulins, both circulating and lymphocyte surface bound. Very few cases of immunoglobulin Kappa or Lambda light chain defects have been reported. Furthermore, the genetic basis of this defect has been extensively explored only in a single case. We report a case of a patient suffering of serious recurrent bacterial infections, which was caused by a very rare form of immunoglobulin disorder, consisting of a pure defect of Kappa light chain. We evaluated major serum immunoglobulin concentrations, as well as total and free Kappa and Lambda light chain concentrations. Lymphocyte phenotyping was also performed and finally we tested the Kappa chain VJ rearrangement as well as the constant Kappa region sequence. Studies performed on VJ rearrangement showed a polyclonal genetic arrangement, whereas the gene sequencing for the constant region of Kappa chain showed a homozygous T to G substitution at the position 1288 (rs200765148). This mutation causes a substitution from Cys to Gly in the protein sequence and, therefore, determines the abnormal folding of the constant region of Kappa chain. We suggest that this defect could lead to an effective reduction of the variability of total antibody repertoire and a consequent defect of an apparently normal immunoglobulin response to common antigens.

  20. The use of immunoglobulin light chain assays in the diagnosis of paraprotein-related kidney disease

    PubMed Central

    Yadav, Punit; Leung, Nelson; Sanders, Paul W.; Cockwell, Paul

    2016-01-01

    Kidney involvement is common in paraprotein-related diseases. A diversity of clinical presentations and histopathological features can occur secondary to tissue injury caused by precipitation or deposition of a clonal immunoglobulin, usually an immunoglobulin light chain. The paraprotein is either produced by multiple myeloma or by a clone of B-cell lineage that does not fulfill diagnostic criteria for multiple myeloma. The recent introduction of serum immunoglobulin free light chain assays, which accurately quantify both light chain isotypes to produce a ratio that indicates the presence or absence of a light chain paraprotein, is a major clinical development. However, as the interpretation of the assay can be challenging, the aim of this review is to clarify the role of serum and urinary light chain assays in the screening and diagnosis of paraprotein-related kidney disease. PMID:25296094

  1. A second immunoglobulin light chain isotype in the rainbow trout.

    PubMed

    Partula, S; Schwager, J; Timmusk, S; Pilström, L; Charlemagne, J

    1996-01-01

    A novel immunoglobulin (Ig) light chain isotype, termed IgL2, has been isolated from trout lymphoid tissues both by reverse transcription - polymerase chain reaction (PCR) and screening of cDNA libraries. The CL domain of the new isotype shares only 29% residues with a recently cloned trout IgL isotype, termed IgL1, which has some similarities to Ckappa and Clambda isotype domains of several vertebrate species. Using anchored PCR, a VL element rearranged to CL2 was isolated. It is a member of a new VL family (VL2) of which four members were sequenced. These differ in the sequence of CDR1 and CDR2 but are remarkably similar in CDR3, i. e., at the junction between VL and JL segments. VL elements are rearranged to novel JL elements which differ from those described for VL1-CL1 rearrangements. Two cDNA clones contained JL-CL2 segments but no VL segments. The JL segments were preceded by typical rearrangements signal sequences [RSS, nonamer-23 base pair (bp) spacer-heptamer]. Further upstream of RSS were located two to three near identical 53 bp repeats, each of which included a 16 bp sequence similar to KI and KII sequences located at similar places in human and mouse Jk1 genes. These sequences are believed to act as binding sites for the protein KLP, which could be a transcriptional factor involved in the synthesis of germline Jk transcripts. Their phylogenic conservation in vertebrates suggests that they have an important role in B-cell differentiation. Remarkably, an RNA species of about 0.7 kilobase is the predominant IgL mRNA in trout spleen and coincides in size with JLCL2 transcripts. Genomic DNA blot analysis indicates that the trout L2 locus has a cluster-like organization similar to the trout L1 locus and the IgL locus of several teleost fish. A phylogenic analysis of VL2 and CL2 corroborates their low similarity to other vertebrate IgL chains and suggests an ancient diversification of the IgL locus. PMID:8881036

  2. Adult Fanconi syndrome with monoclonal abnormality of immunoglobulin light chain

    PubMed Central

    Harrison, J. F.; Blainey, J. D.

    1967-01-01

    Two adult cases of the Fanconi syndrome are described, in each of which there was abnormal urinary excretion of immunoglobulin κ-chain. The significance of this finding is discussed in relation to the recognized association between multiple myeloma and the Fanconi syndrome. Images PMID:6016886

  3. The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

    SciTech Connect

    Pozo-Yauner, Luis del; Wall, Jonathan S.; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L.; Pérez Carreón, Julio I.; and others

    2014-01-10

    Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.

  4. Systemic immunoglobulin light-chain amyloidosis presenting hematochezia as the initial symptom.

    PubMed

    Kon, Tetsuo; Nakagawa, Naoki; Yoshikawa, Fumitsugu; Haba, Kazunao; Kitagawa, Nagako; Izumi, Michihiro; Kumazaki, Setsuo; Ishida, Satoshi; Aikawa, Ryuichi

    2016-08-01

    Immunoglobulin light-chain (AL) amyloidosis is characterized by the deposition of insoluble fibrils composed of immunoglobulin light chains secreted by monoclonal plasma cells. Given the recent advances in the therapy of AL amyloidosis, it is important to diagnose this disease as early as possible. Herein, we describe the case of a 62-year-old man with hepatitis C virus (HCV)-related cirrhosis presenting with hematochezia. Colonoscopy showed multiple submucosal hematomas within the region ranging from the transverse colon to the sigmoid colon. Kappa immunoglobulin light-chain amyloid deposition was also detected. Bone marrow examination revealed a monoclonal abnormal plasma cell population. Thus, the patient was diagnosed with systemic immunoglobulin light-chain amyloidosis. The hematochezia was conservatively managed. However, because of liver failure caused by liver cirrhosis, the patient developed massive pleural effusion and died of respiratory failure. Postmortem examination revealed amyloid deposition in the esophagus, stomach, duodenum, ileum, descending colon, pancreas, heart, and lung. In these organs, amyloid deposition was limited to the vascular wall. We concluded that AL amyloidosis can present hematochezia arising from submucosal hematoma in the large colon before other systemic symptoms appear.

  5. Systemic immunoglobulin light-chain amyloidosis presenting hematochezia as the initial symptom.

    PubMed

    Kon, Tetsuo; Nakagawa, Naoki; Yoshikawa, Fumitsugu; Haba, Kazunao; Kitagawa, Nagako; Izumi, Michihiro; Kumazaki, Setsuo; Ishida, Satoshi; Aikawa, Ryuichi

    2016-08-01

    Immunoglobulin light-chain (AL) amyloidosis is characterized by the deposition of insoluble fibrils composed of immunoglobulin light chains secreted by monoclonal plasma cells. Given the recent advances in the therapy of AL amyloidosis, it is important to diagnose this disease as early as possible. Herein, we describe the case of a 62-year-old man with hepatitis C virus (HCV)-related cirrhosis presenting with hematochezia. Colonoscopy showed multiple submucosal hematomas within the region ranging from the transverse colon to the sigmoid colon. Kappa immunoglobulin light-chain amyloid deposition was also detected. Bone marrow examination revealed a monoclonal abnormal plasma cell population. Thus, the patient was diagnosed with systemic immunoglobulin light-chain amyloidosis. The hematochezia was conservatively managed. However, because of liver failure caused by liver cirrhosis, the patient developed massive pleural effusion and died of respiratory failure. Postmortem examination revealed amyloid deposition in the esophagus, stomach, duodenum, ileum, descending colon, pancreas, heart, and lung. In these organs, amyloid deposition was limited to the vascular wall. We concluded that AL amyloidosis can present hematochezia arising from submucosal hematoma in the large colon before other systemic symptoms appear. PMID:27318996

  6. Immunoglobulin heavy chain/light chain pairs (HLC, Hevylite™) assays for diagnosing and monitoring monoclonal gammopathies.

    PubMed

    Kraj, Maria

    2014-01-01

    Immunofixation (IFE) is a standard method for detecting monoclonal immunoglobulins and characterizing its isotype. Recently clonality can also be determined by using immunoglobulin (Ig) heavy chain/light chain immunoassays - HLC, HevyliteTM. HLC separately measures in pairs light chain types of each intact Ig class generating ratios of monoclonal Ig/uninvolved polyclonal Ig concentrations. Studies have shown that HLC and IFE are complementary methods. HLC assays quantify monoclonal proteins and identify monoclonality. It is possible to predict prognosis in multiple myeloma and to monitor response to treatment using HLC ratio. HLC ratio may serve as a parameter for myeloma induced immunoparesis and serve as a new marker for validating remission depth and relapse probabilities.

  7. A novel monoclonal antibody against the constant region of goose immunoglobulin light chain.

    PubMed

    Guo, Yongli; Gao, Mingchun; Ma, Bo; Sheng, Qiaoling; Wang, Qian; Liu, Dandan; Wang, Junwei

    2014-04-01

    A monoclonal antibody (MAb) against the antigenic determinant of the constant region of goose immunoglobulin light chain (GoIgCL) was produced and characterized for the first time here. Goose immunoglobulin (Ig) in serum was purified by immunoaffinity chromatography and the resulting protein was used as immunogen to immunize BALB/c mice. At the same time, the GoIgCL gene was expressed and purified as the screening antigen for selecting MAb against GoIgCL. One hybridoma that produces antibodies against GoIgCL was selected by indirect ELISA. Then the characterization of the MAb was analyzed by ELISA, Western blot, and flow cytometry. It was found to be IgG1 with κ light chain; the MAB has high specificity to Ig in goose serum, bile, and B lymphocytes from peripheral blood, reacts only with the light chain of goose Ig, and can distinguish Ig from other birds. Therefore, the MAb generated in this study can be used as a specific reagent for detection of goose disease-specific antibodies and as a powerful tool for basic immunology research on geese.

  8. Immunoglobulin Light-Chain Amyloidosis: From Basics to New Developments in Diagnosis, Prognosis and Therapy.

    PubMed

    Muchtar, Eli; Buadi, Francis K; Dispenzieri, Angela; Gertz, Morie A

    2016-01-01

    Immunoglobulin amyloid light-chain (AL) amyloidosis is the most common form of systemic amyloidosis, where the culprit amyloidogenic protein is immunoglobulin light chains produced by marrow clonal plasma cells. AL amyloidosis is an infrequent disease, and since presentation is variable and often nonspecific, diagnosis is often delayed. This results in cumulative organ damage and has a negative prognostic effect. AL amyloidosis can also be challenging on the diagnostic level, especially when demonstration of Congo red-positive tissue is not readily obtained. Since as many as 31 known amyloidogenic proteins have been identified to date, determination of the amyloid type is required. While several typing methods are available, mass spectrometry has become the gold standard for amyloid typing. Upon confirming the diagnosis of amyloidosis, a pursuit for organ involvement is essential, with a focus on heart involvement, even in the absence of suggestive symptoms for involvement, as this has both prognostic and treatment implications. Details regarding initial treatment options, including stem cell transplantation, are provided in this review. AL amyloidosis management requires a multidisciplinary approach with careful patient monitoring, as organ impairment has a major effect on morbidity and treatment tolerability until a response to treatment is achieved and recovery emerges.

  9. Proliferative glomerulonephritis with monoclonal immunoglobulin deposition disease: The utility of routine staining with immunoglobulin light chains

    PubMed Central

    Gowda, K. K.; Nada, R.; Ramachandran, R.; Joshi, K.; Tewari, R.; Kohli, H. S.; Jha, V.; Gupta, K. L.

    2015-01-01

    Proliferative glomerulonephritis occurring as a consequence of monoclonal glomerular deposits of IgG is uncommon. It is a form of renal involvement in monoclonal gammopathy that mimics immune complex glomerulonephritis. Here, we report the first series of proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) from the Indian subcontinent highlighting use of light chain immunofluorescence (IF) in routine renal biopsy interpretation. We retrieved 6 patients diagnosed as proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) out of 160 biopsies (3.7%) with membranoproliferative patterns over 5 1/2 years (2009–2014), one of whom had recurrence 6 months post-renal transplant. Four (67%) patients presented with rapidly progressive renal failure and two (33%) with nephrotic syndrome. None of these patients had overt multiple myeloma. The predominant histologic pattern was membranoproliferative with all the biopsies showing IgG3 Kappa deposits on IF. The deposits were primarily subendothelial on electron microscopy. PMID:26664209

  10. Molecular characterization of the immunoglobulin light chain variable region repertoire of human autoantibodies

    SciTech Connect

    Victor, K.D.

    1992-01-01

    The molecular structures of the light chain variable regions encoding human autoantibodies have been studied in detail. The variable region repertoire among this group of antibodies is diverse. There is no evidence for preferential utilization of specific V[sub L] gene families or over-representation of certain V[sub L] gene segments in autoantibodies. Many autoreactive antibodies utilize direct copies of known germline gene segments with little evidence of somatic mutation, supporting the conclusion that at least some germline gene segments encode autoreactivity. Additionally, the structures of several autoantibodies are clearly the product of somatic mutation. Lastly, affinity maturation has been demonstrated in two clonally related IgM rheumatoid factors suggestive of an antigen driven response. The heterogeneity of the V[sub L] region repertoire in human autoantibodies challenges evidence in the literature suggesting that the majority of human autoantibodies utilize the same or closely related germline gene segments with no evidence of somatic mutation. In addition, this study has documented that variation in the length of the light chain is a common feature in human antibodies. Length variation is confined to the V[sub k]-J[sub k] joint of CDR3 and occurs in all V[sub k] gene families. Analysis of the structures of the V[sub k]-J[sub k] joints suggests that both germline derived and non-germline encoded nucleotides (N-segments), probably the result of terminal deoxynucleotidyl transferase activity, contribute to the junctional diversity of the immunoglobulin light chain variable region. Thus, length variation at the V[sub L]-J[sub L] joint is a frequent event having the potential to expand the diversity of the antibody molecule.

  11. Light chain amyloidosis of the urinary bladder. A site restricted deposition of an externally produced immunoglobulin

    PubMed Central

    Livneh, A; Shtrasburg, S; Martin, B; Baniel, J; Gal, R; Pras, M

    2001-01-01

    Aims—To identify the amyloid protein in a patient with amyloidosis localised to the urinary bladder, and to see whether subtyping of the protein by sequence analysis increases the understanding of the selection of the urinary bladder as the site of amyloid deposition. Methods—A patient with gross haematuria and a congophilic mass in his urinary bladder was evaluated further. Characterisation of the amyloid protein was performed using conventional histological and immunohistochemical methods. Determination of the N-terminal amino acid sequence of the amyloid protein was performed using protein sequencers. Results—The patient's history, physical examination, and laboratory evaluation excluded the involvement of other organs, justifying a diagnosis of amyloidosis localised to the urinary bladder. Histological and immunological studies showed that the amyloid protein deposited in the urinary bladder of the patient was probably of the amyloid light chain type. No plasma cells or lymphocytes were seen in sections of the urinary bladder and lower ureter adjacent to the amyloid deposits. Molecular analysis showed the sequence NFMLTQPHSISGSPG, which assigned the amyloid protein to either the VλI or the VλVI immunoglobulin (Ig) light chain families. Conclusions—The findings suggest that the amyloid protein in this patient originated outside the urinary bladder. The heterogeneity of the Ig proteins in known cases of amyloidosis of the lower urinary tract suggests that the amino acid residues, which determine the Vλ subtyping, have no major role in restricting the deposited protein to the urinary bladder. Key Words: primary amyloidosis • urinary bladder • λ light chain • amino acid sequence PMID:11729210

  12. Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173.

    PubMed

    Steinmetz, M; Zachau, H G; Mach, B

    1979-07-25

    The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images

  13. [The role of the assessment of heavy/light chain pairs of immunoglobulin in monoclonal gammopathies].

    PubMed

    Ščudla, Vlastimil; Pika, Tomáš; Minařík, Jiří

    2015-01-01

    The aim of the paper is to inform about the contribution of novel, highly sensitive analytic technique for the assessment of serum immunoglobulins (Hevylite), enabling separate quantitative assessment of heavy/light chain pairs of immunoglobulin (HLC), i. e. the monoclonal ("involved") and polyclonal ("noninvolved") isotype including their ratio (HLC-r) in monoclonal gammopathies. We particularly target the characteristics of this technique, the compari-son of its clinical contribution with standard methods used in the diagnostics, course and the detection of relapse and progression of the disease, as well as the stratification, assessment of therapeutic outcome and prognosis in monoclonal gammopathy of undetermined significance, multiple myeloma, Waldenström´s macroglobulinemia, systemic AL-amyloidosis and some non-Hodgkin lymphomas. Present results show that in comparison with existing routinely used techniques the Hevylite method enriches clinical practice with the assessment of serum levels of "uninvolved" Ig. It enables the evaluation of the depth of "immunoparesis", and the determination of HLC-r index that is needful for the stratification of MM into "risk cohorts". It also contributes to prognostic assessment and improvement of the evaluation of the depth of therapeutic response. In MGUS individuals the HLC-r index provides information about the risk of malignant transformation. We await the results of ongoing validation studies that are expected to provide specific indications for Hevylite technique for MG in routine practice.

  14. Production and characterization of monoclonal antibodies specific for kappa and. gamma. light chain types of porcine immunoglobulins

    SciTech Connect

    McCauley, I.; Kim, Y.B.

    1986-03-05

    It has been difficult to raise specific antisera to the light chain types of pigs because of the difficulty in isolating sufficient pure material from polyclonal immunoglobulin. The authors have taken an approach based upon the characterization of a number of monoclonal antibodies (MoAb) raised against porcine IgG in order to obtain antisera specific for light chain types. Spleen cells from mice immunized with porcine IgG were fused with myeloma P3x63-Ag 653. Hybridomas were screened by an ELISA technique against pure porcine light chains coated on microtiter plates. Five clones specific for light chains were isolated. MoAb from these clones have been characterized by sequential immunoprecipitation of /sup 125/I labelled light chains. The pattern of reactivities show that the MoAb can be classified into two mutually exclusive groups, each of which precipitate approximately equal amounts of the labelled light chains. The type specificity of these groups has been determined by utilizing the cross-reaction between anti-human kappa and ..gamma.. with porcine light chains and the groups of MoAb in sequential immunoprecipitations. The MoAb were used in an immunofluorescence study of porcine B lymphocytes. The anti-..gamma.. MoAb stained 57% and the anti-kappa, 43% of total B lymphocytes.

  15. Effect of specimen type on free immunoglobulin light chains analysis on the Roche Diagnostics cobas 8000 analyzer.

    PubMed

    Nelson, Louis S; Steussy, Bryan; Morris, Cory S; Krasowski, Matthew D

    2015-01-01

    The measurement of free immunoglobulin light chains is typically performed on serum; however, the use of alternative specimen types has potential benefits. Using the Freelite™ kappa and lambda free light chains assay on a Roche Diagnostics cobas 8000 c502 analyzer, we compared three specimen types (serum, EDTA-plasma and lithium heparin plasma separator gel-plasma) on 100 patients. Using Deming regression and eliminating outliers (limiting data to light chain concentrations below 400 mg/L), the three specimen types showed comparable results for kappa light chain concentration, lambda light chain concentration, and kappa/lambda ratio with slopes close to 1.0 and y-intercepts close to zero. EDTA-plasma showed slightly more positive bias relative to serum than lithium heparin. Analysis using EDTA-plasma and lithium heparin plasma showed comparable linearity, precision, and temperature stability. A single sample showing hook effect (not in the comparison set) gave comparable results using either plasma specimen type. For the Freelite™ kappa and lambda free light chains assay, both EDTA-plasma or lithium heparin-plasma can serve as acceptable substitutes for serum, at least for the Roche cobas 8000 analyzer. PMID:26682113

  16. Structural Characterization of the Partially Folded Intermediates of An Immunoglobulin Light Chain Leading to Amyloid Fibrillation And Amorphous Aggregation

    SciTech Connect

    Qin, Z.; Hu, D.; Zhu, M.; Fink, A.L.; /UC, Santa Cruz

    2007-07-12

    Immunoglobulin light chain deposition diseases involve various types of extracellular deposition of light chain variable domains, including amyloid fibrils and amorphous deposits. The decreased thermodynamic stability of the light chain is believed to be the major factor leading to fibrillation. However, the differences in the nature of the deposits among the light chain deposition diseases raise the question of whether the mechanisms leading to fibrillar or amorphous aggregation is different. In this study, we generated two partially folded intermediates of the light chain variable domain SMA in the presence of guanidine hydrochloride (GuHCl) and characterized their conformations. The more unfolded intermediate formed fibrils most rapidly, while the more native-like intermediate predominantly led to amorphous deposits. The results also show that the monomeric, rather than the dimeric state, was critical for fibrillation. The data also indicate that fibril elongation involves addition of a partially unfolded intermediate, rather than the native state. We postulate that a more highly unfolded intermediate is more suited to undergo the topological rearrangements necessary to form amyloid fibrils than a more structured one and that this also correlates with increased destabilization. In the case of light chain aggregation, it appears that more native-like intermediate conformations are more prone to form amorphous deposits.

  17. Immunoparesis status in immunoglobulin light chain amyloidosis at diagnosis affects response and survival by regimen type

    PubMed Central

    Muchtar, Eli; Dispenzieri, Angela; Kumar, Shaji K.; Dingli, David; Lacy, Martha Q.; Buadi, Francis K.; Hayman, Suzanne R.; Kapoor, Prashant; Leung, Nelson; Chakraborty, Rajshekhar; Russell, Stephen; Lust, John A.; Lin, Yi; Go, Ronald S.; Zeldenrust, Steven; Kyle, Robert A.; Rajkumar, S. Vincent; Gertz, Morie A.

    2016-01-01

    Clinical tools to guide in the appropriate treatment selection in immunoglobulin light chain (AL) amyloidosis are not well developed. We evaluated the response and outcome for various regimens at first-line treatment (n=681) and first progression (n=240) stratified by the immunoparesis status at diagnosis. Immunoparesis was assessed by the average relative difference of the uninvolved immunoglobulins, classifying patients into a negative average relative difference (i.e. significant immunoparesis) or a positive average relative difference (no/modest immunoparesis). Treatment was categorized as autologous stem cell transplant and four non-transplant regimens (melphalan-based; bortezomib-based, immunomodulatory drug-based and dexamethasone alone). Patients with significant immunoparesis who underwent stem cell transplant had a significantly lower rate of very good partial response or better response (58%), progression-free survival (median 30 months) and overall survival (108 months), compared to those without significant immunoparesis (80%, 127 months, median not reached, respectively; P<0.001 for all comparisons). Among the non-transplant regimens, melphalan resulted in an unfavorable progression-free survival (11 vs. 27 months; P<0.001) and overall survival (30 vs. 74 months; P=0.001) in patients with significant immunoparesis compared to those without significant immunoparesis. In contrast, no significant difference in outcomes between the immunoparesis groups was seen for those treated with bortezomib or immunomodulatory drugs. At first progression, immunoparesis status did not impact response or survival of any regimen. Melphalan at first-line provided poorer outcomes for patients with significant immunoparesis, while bortezomib or immunomodulatory drugs were more likely to overcome the adverse prognosis associated with significant immunoparesis. PMID:27479823

  18. Diagnostic value of immunoglobulin κ light chain gene rearrangement analysis in B-cell lymphomas

    PubMed Central

    KOKOVIC, IRA; NOVAKOVIC, BARBARA JEZERSEK; NOVAKOVIC, SRDJAN

    2015-01-01

    Analysis of the immunoglobulin κ light chain (IGK) gene is an alternative method for B-cell clonality assessment in the diagnosis of mature B-cell proliferations in which the detection of clonal immunoglobulin heavy chain (IGH) gene rearrangements fails. The aim of the present study was to evaluate the added value of standardized BIOMED-2 assay for the detection of clonal IGK gene rearrangements in the diagnostic setting of suspected B-cell lymphomas. With this purpose, 92 specimens from 80 patients with the final diagnosis of mature B-cell lymphoma (37 specimens), mature T-cell lymphoma (26 specimens) and reactive lymphoid proliferation (29 specimens) were analyzed for B-cell clonality. B-cell clonality analysis was performed using the BIOMED-2 IGH and IGK gene clonality assays. The determined sensitivity of the IGK assay was 67.6%, while the determined sensitivity of the IGH assay was 75.7%. The sensitivity of combined IGH+IGK assay was 81.1%. The determined specificity of the IGK assay was 96.2% in the group of T-cell lymphomas and 96.6% in the group of reactive lesions. The determined specificity of the IGH assay was 84.6% in the group of lymphomas and 86.2% in the group of reactive lesions. The comparison of GeneScan (GS) and heteroduplex pretreatment-polyacrylamide gel electrophoresis (HD-PAGE) methods for the analysis of IGK gene rearrangements showed a higher efficacy of GS analysis in a series of 27 B-cell lymphomas analyzed by both methods. In the present study, we demonstrated that by applying the combined IGH+IGK clonality assay the overall detection rate of B-cell clonality was increased by 5.4%. Thus, we confirmed the added value of the standardized BIOMED-2 IGK assay for assessment of B-cell clonality in suspected B-cell lymphomas with inconclusive clinical and cyto/histological diagnosis. PMID:25501347

  19. Purification and characterization of a stimulator of plasmin generation from the antiangiogenic agent Neovastat: identification as immunoglobulin kappa light chain.

    PubMed

    Boivin, Dominique; Provençal, Mathieu; Gendron, Sébastien; Ratel, David; Demeule, Michel; Gingras, Denis; Béliveau, Richard

    2004-11-15

    We have recently shown that Neovastat, an antiangiogenic extract from shark cartilage, stimulates the in vitro activation of plasminogen by facilitating the tissue-type plasminogen activator (tPA)-dependent conversion of plasminogen to plasmin. In this report, we describe the purification and characterization of the stimulatory molecules. Neovastat was subjected to a three-step purification procedure including gel filtration, preparative isoelectric focusing, and preparative SDS-PAGE. Two 28-kDa proteins with pIs of approximately 4.5 and 6.5 were purified to apparent homogeneity and identified as immunoglobulin (Ig) kappa light chains by N-terminal microsequencing. Ig light chains do not directly stimulate the activity of tPA or plasmin, suggesting a mechanism of action involving an interaction with plasminogen. Kinetic analysis showed that both Ig light chains accelerate the in vitro tPA-dependent conversion of plasminogen in plasmin by increasing the affinity of tPA for plasminogen by 32- and 38-fold (Km decrease from 456 nM to 12-14 nM). Shark Ig light chains also stimulated the degradation of fibrin by the tPA/plasminogen system in an in vitro assay. A direct interaction between Ig light chains and plasminogen (KA=4.0-5.5 x 10(7) M(-1); KD=18-25 nM) and with tPA (KA=2.8 x 10(7) M(-1); KD=36 nM) was demonstrated using real time binding measured by surface plasmon resonance. Ig light chain is the first molecule associated with the antiangiogenic activity of Neovastat to be purified and identified. PMID:15488468

  20. Long term outcomes of cardiac transplant for immunoglobulin light chain amyloidosis: The Mayo Clinic experience

    PubMed Central

    Grogan, Martha; Gertz, Morie; McCurdy, Arleigh; Roeker, Lindsey; Kyle, Robert; Kushwaha, Sudhir; Daly, Richard; Dearani, Joseph; Rodeheffer, Richard; Frantz, Robert; Lacy, Martha; Hayman, Suzanne; McGregor, Christopher; Edwards, Brooks; Dispenzieri, Angela

    2016-01-01

    AIM: To determine the outcome of orthotopic heart transplantation (OHT) in immunoglobulin light chain (AL) amyloidosis. METHODS: The medical records of patients with AL who underwent orthotopic heart transplantation at the Mayo Clinic in Rochester Minnesota from 1992 to 2011 were reviewed. Patients met at least one of the following at: New York Heart Association class IV heart failure, ventricular thickness > 15 mm, ejection fraction < 40%. Selection guidelines for heart transplant included age < 60 years, absence of multiple myeloma and significant extra-cardiac organ involvement. Baseline characteristics including age, gender, organ involvement, and New York Heart Association functional class were recorded. Laboratory data, waiting time until heart transplant, and type of treatment of the underlying plasma cell disorder were recorded. Survival from the time of OHT was calculated using Kaplan-Meier survival curves. Survival of patients undergoing OHT for AL was compared to that of non-amyloid patients undergoing OHT during the same time period. RESULTS: Twenty-three patients (median age 53 years) with AL received OHT. There were no deaths in the immediate perioperative period. Twenty patients have died post OHT. For the entire cohort, the median overall survival was 3.5 years (95%CI: 1.2, 8.2 years). The 1-year survival post OHT was 77%, the 2-year survival 65%, and the 5-year survival 43%. The 5-year survival for non-amyloid patients undergoing OHT during the same era was 85%. Progressive amyloidosis contributed to death in twelve patients. Of those without evidence of progressive amyloidosis, the cause of death included complications of autologous hematopoietic stem cell transplantation for 3 patients, post-transplant lymphoproliferative disorder for 2 patients; and for the remaining one death was related to each of the following causes: acute rejection; cardiac vasculopathy; metastatic melanoma; myelodysplastic syndrome; and unknown. Eight patients had

  1. Immunoglobulin light chain immunohistochemistry revisited, with emphasis on reactive follicular hyperplasia versus follicular lymphoma.

    PubMed

    Weiss, Lawrence M; Loera, Sofia; Bacchi, Carlos E

    2010-05-01

    The identification of monotypic light chains is an important adjunct to the diagnosis of B-cell lymphoma, yet to reliably perform it on formalin-fixed paraffin sections is often difficult. We have evaluated a new set of monoclonal antibodies to kappa and lambda light chains that are reactive in paraffin sections. In reactive lymphoid tissues, polytypic staining was noted in greater than 95% of cases, with strong staining of plasma cells, moderate staining of the follicular dendritic cell network, and weak staining of mantle zone cells. Strong staining of the appropriate light chain was seen in each of the 7 cases of multiple myeloma. In a series of 58 cases of B-cell lymphoma, correlation between the results of immunohistochemistry and flow cytometry was obtained in 36 cases (62%), including 32 cases (21 kappa and 11 lambda) in which a single light chain was expressed. Monotypic staining was also seen in 6 additional cases (10%) in which flow cytometry was negative. Thirty of 46 cases (65%) of follicular lymphoma showed monotypic light chain expression, in contrast to 64 of 67 cases (95%) of reactive lymphoid hyperplasia, which showed polytypic light chain expression. These antibodies may provide an effective adjunct to the diagnosis of B-cell lymphoma in routine diagnostic work.

  2. A molecular model for self-assembly of amyloid fibrils: Immunoglobulin light chains

    SciTech Connect

    Stevens, F.J.; Myatt, E.A.; Westholm, F.A.

    1995-08-29

    The formation and pathological deposition of amyloid fibrils are defining features of many acquired and inherited disorders, including primary or light-chain-associated amyloidosis, Alzheimer`s disease, and adult-onset diabetes. No pharmacological methods exist to block this process or to effect the removal of fibrils from tissue, and thus, little can be done to prevent organ failure and ultimate death that result from deposition of amyloid. Knowledge of the pathogenesis, treatment, or prevention of these presently incurable diseases is limited due to the relative paucity of information regarding the biophysical basis of amyloid formation. Antibody light chains of different amino acid sequence show differential amyloid-forming tendencies and, as such, can provide insight into the structural organization of amyloid fibrils as well as into basic mechanisms of protein self-assembly. We have compared primary structures of 180 human monoclonal light chains and have identified particular residues and positions within the variable domain that differentiate amyloid-from nonamyloid-associated proteins. We propose a molecular model that accounts for amyloid formation by antibody light chains and might also have implications for other forms of amyloidosis. 24 refs., 2 figs., 1 tab.

  3. Suppression of uninvolved immunoglobulins defined by heavy/light chain pair suppression is a risk factor for progression of MGUS

    PubMed Central

    Katzmann, JA; Clark, R; Kyle, RA; Larson, DR; Therneau, TM; Melton, LJ; Benson, JT; Colby, CL; Dispenzieri, A; Landgren, O; Kumar, S; Bradwell, AR; Cerhan, JR; Rajkumar, SV

    2013-01-01

    We hypothesized that the suppression of uninvolved immunoglobulin in monoclonal gammopathy of undetermined significance (MGUS) as detected by suppression of the isotype-specific heavy and light chain (HLC-pair suppression) increases the risk of progression to malignancy. This approach required quantitation of individual heavy/light chains (for example, IgGλ in IgGκ MGUS patients). Of 1384 MGUS patients from Southeastern Minnesota seen at the Mayo Clinic from 1960 to 1994, baseline serum samples obtained within 30 days of diagnosis were available in 999 persons. We identified HLC-pair suppression in 27% of MGUS patient samples compared with 11% of patients with suppression of uninvolved IgG, IgA or IgM. HLC-pair suppression was a significant risk factor for progression (hazard ratio (HR), 2.3; 95% confidence interval (CI) 1.5–3.7; P<0.001). On multivariate analysis, HLC-pair suppression was an independent risk factor for progression to malignancy in combination with serum M-spike size, heavy chain isotype and free light chain ratio (HR, 1.8; 95% CI, 1.1–3.00; P = 0.018). The finding that HLC-pair suppression predicts progression in MGUS and occurs several years before malignant transformation has implications for myeloma biology. PMID:22781594

  4. Restricted Immunoglobulin Variable Region (Ig V) Gene Expression Accompanies Secondary Rearrangements of Light Chain Ig V Genes in Mouse Plasmacytomas

    PubMed Central

    Diaw, Lena; Siwarski, David; Coleman, Allen; Kim, Jennifer; Jones, Gary M.; Dighiero, Guillaume; Huppi, Konrad

    1999-01-01

    The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl. Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (VL) and heavy (VH) variable region genes to express a characteristic monoclonal antibody. In this study, we have examined by enzyme-linked immunosorbent assay five antibodies secreted by silicone-induced mouse plasmacytomas using a broader panel of antigens including actin, myosin, tubulin, single-stranded DNA, and double-stranded DNA. We have determined the Ig heavy and light chain V gene usage in these same plasmacytomas at the DNA and RNA level. Our studies reveal: (a) antibodies secreted by plasmacytomas bind to different antigens in a manner similar to that observed for natural autoantibodies; (b) the expressed Ig heavy genes are restricted in V gene usage to the VH-J558 family; and (c) secondary rearrangements occur at the light chain level with at least three plasmacytomas expressing both κ and λ light chain genes. These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes. PMID:10562316

  5. Phage Display and Peptide Mapping of an Immunoglobulin Light Chain Fibril-Related Conformational Epitope†

    PubMed Central

    O’Nuallain, Brian; Allen, Amy; Ataman, Demet; Weiss, Deborah T.; Solomon, Alan; Wall, Jonathan S.

    2008-01-01

    Amyloid fibrils and partially unfolded intermediates can be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that mAb 11-1F4, generated by immunizing mice with a thermally denatured variable domain (VL) fragment of the human κ4 Bence Jones protein Len, bound to a non-native conformational epitope located within the N-terminal 18 residues of fibrillar, as well as partially denatured, Ig light chains (O’Nuallain B. et al. (2006) Biochemistry 46, 1240–247). To define further the antibody binding site, we used random peptide phage display and epitope mapping of VL Len using wild-type and alanine-mutated Len peptides where it was shown that the antibody epitope was reliant on up to 10 of the first 15 residues of protein Len. Comparison of Vκ and Vλ N-terminal germline consensus sequences with protein Len and 11-1F4-binding phages indicated that this antibody’s cross-reactivity with light chains was related to an invariant proline at position(s) 7 and/or 8, bulky hydrophobic residues at positions 11 and 13, and additionally, to the ability to accommodate amino acid diversity at positions 1–4. Sequence alignments of the phage peptides revealed a central proline, often flanked by aromatic residues. Taken together, these results have provided evidence for the structural basis of the specificity of 11-1F4 for both κ and λ light chain fibrils. We posit that the associated binding site involves a rare type VI β-turn or touch-turn that is anchored by a cis-proline residue. The identification of an 11-1F4-related mimotope should facilitate development of pan-light chain fibril-reactive antibodies that could be used in the diagnosis and treatment of patients with AL amyloidosis. PMID:17944486

  6. Immunoglobulin heavy/light chain analysis enhances the detection of residual disease and monitoring of multiple myeloma patients

    PubMed Central

    Batinić, Josip; Perić, Zinaida; Šegulja, Dragana; Last, James; Prijić, Sanja; Dubravčić, Klara; Volarić, Lidija; Sertić, Dubravka; Radman, Ivo; Bašić-Kinda, Sandra; Matišić, Danica; Batinić, Drago; Labar, Boris; Nemet, Damir

    2015-01-01

    Aim To evaluate the clinical utility of incorporating a novel heavy/light chain immunoassay (HLC) into the existing methods for the assessment of multiple myeloma (MM) patients. Methods Convenience sera samples from 90 previously treated IgG and IgA MM patients in different disease stages were analyzed. The study was conducted in Clinical Hospital Center Zagreb between 2011 and 2013. The collected sera were analyzed by standard laboratory techniques (serum protein electrophoresis, quantification of total immunoglobulins, serum immunofixation, serum free light chain [FLC] assay) and HLC assay. Results HLC ratios outside the normal range were found in 58 of 90 patients, including 28 out of 61 patients with total immunoglobulin measurements within the normal range and 5 out of 23 patients in complete response. Both elevated HLC isotype level and abnormal HLC ratio correlated with the parameters of tumor burden, including percentage of plasma cells in the bone marrow (P < 0.001 and P = 0.002, respectively) and an abnormal serum FLC ratio (for both P < 0.001). In addition, abnormal HLC isotype level correlated with serum beta-2-microglobulin level (P = 0.038). In terms of prognosis, abnormal HLC isotype level and abnormal HLC ratio were significantly associated with shorter overall survival (P < 0.001 and P = 0.002, respectively). Interestingly, suppression of the uninvolved (polyclonal) isotype pair, but not other non-myeloma immunoglobulin isotypes, was also associated with a shorter overall survival (P = 0.021). In a multivariate analysis, an abnormal HLC ratio and β2-microglobulin level >3.5mg/L were independent risk factors for survival. Conclusion The new HLC assay has greater sensitivity in detecting monoclonal protein, correlates with tumor burden markers, and affects patients' outcome. PMID:26088851

  7. Inhibition of pathologic immunoglobulin free light chain production by small interfering RNA molecules

    PubMed Central

    Phipps, Jonathan E.; Kestler, Daniel P.; Foster, James S.; Kennel, Stephen J.; Donnell, Robert; Weiss, Deborah T.; Solomon, Alan; Wall, Jonathan S.

    2010-01-01

    Objectives Morbidity and mortality occurring in patients with multiple myeloma, AL amyloidosis, and light chain deposition disease can result from the pathologic deposition of monoclonal Ig light chains (LCs) in kidneys and other organs. To reduce synthesis of such components, therapy for these disorders typically has involved anti-plasma cell agents; however, this approach is not always effective and can have adverse consequences. We have investigated another means to achieve this objective; namely, RNA interference (RNAi). Materials and Methods SP2/O mouse myeloma cells were stably transfected with a construct encoding a λ6 LC (Wil) under control of the CMV promoter, while λ2-producing myeloma cell line RPMI 8226 was purchased from the ATCC. Both were treated with small interfering RNA (siRNA) directed specifically to the V, J, or C portions of the molecules and then analyzed by ELISA, flow cytometry and real time PCR. Results Transfected cells were found to constitutively express detectable quantities of mRNA and protein Wil and, after exposure to siRNAs, an ~40% reduction in mRNA and LC production was evidenced at 48 hours. An even greater effect was seen with the 8226 cells. Conclusion Our results have shown that RNAi can markedly reduce LC synthesis and provide the basis for testing the therapeutic potential of this strategy using in vivo experimental models of multiple myeloma. PMID:20637260

  8. [EXPRESSION OF THE LIGHT CHAINS OF IMMUNOGLOBULINS IN NORMAL B-CELLS AND SOME B-CELL LYMPHOMAS].

    PubMed

    Khudoleeva, O A; Vorobjev, I A

    2015-01-01

    The quantitative method of determining the level of expression of immunoglobulin light chains on uncompensated data was suggested and used to examine disorders in light chain expression in various B-cell tumors. The average level of expression of the lambda isotype was 4 times higher than the level of expression of kappa isotype. The level of surface and cytoplasmic expression of LC IG varied within wide limits for different people, but there was a high degree of correlation between the levels of expression of kappa and lambda isotypes LC IG as well as between expression of the surface and cytoplasmic forms of each in isotype the same individual. In the majority of B-cell non-Hodgkin's lymphomas correlation between the expression of LC IG on the surface and in the cytoplasm of the cells was diminished. Expression of LC IG in CLL was significantly reduced on the surface of the cells and to a lesser extent--in the cytoplasm. In the case of marginal zone cell lymphoma, LC IG expression level was reduced on the surface of circulating cells and to a lesser extent--in the cytoplasm. In the case of mantle cell lymphoma and DLBCL, expression level of LC IG on the cell surface and in the cytoplasm was the same as in normal B-cells. However, in some cases DLBCL, no LC IG was expressed both on the surface and in the cytoplasm. PMID:26863766

  9. MRC OX-2 antigen: a lymphoid/neuronal membrane glycoprotein with a structure like a single immunoglobulin light chain.

    PubMed Central

    Clark, M J; Gagnon, J; Williams, A F; Barclay, A N

    1985-01-01

    The MRC OX-2 antigen is a rat cell surface glycoprotein of mol. wt. 41 000-47 000 found on neurones, thymocytes, B cells, follicular dendritic cells and endothelium. We now report the amino sequence for this antigen as deduced from the nucleotide sequence of cDNA clones detected by use of an oligonucleotide probe. The sequence contains 248 amino acid residues of which 202 residues are likely to be outside the cell with two domains that show homology with immunoglobulins. The N-terminal domain fits best with Ig V domains and Thy-1 antigen while the C-terminal part is like an Ig C domain. Thus the structure overall is similar to an Ig light chain or the T cell receptor beta chain. Three glycosylation sites are identified on each of the MRC OX-2 antigen domains. Images Fig. 1. Fig. 2. PMID:2862025

  10. Molecular characterization and expression analysis of four isotypes of immunoglobulin light chain genes in orange-spotted grouper, Epinephelus coioides.

    PubMed

    Wu, Ming-Shan; Cheng, Chao-An; Lin, Chih-Hung; Lee, Chiou-Yueh; Tseng, Shih-Jou; Tzeng, Chyng-Shyan; Chang, Chi-Yao

    2013-03-01

    To date, many immunoglobulin (Ig) genes have been identified in diverse teleost species, but the contributions of different types of light chain (IgL) to the immune response remain unclear. Screening of a stimulated kidney cDNA library from orange-spotted grouper (Osg, Epinephelus coioides) resulted in the identification of 26 full Ig light chain (OsgIgL) coding sequences. These 26 OsgIgLs encoded peptides from 235 to 248 amino acid residues and could be grouped into five variable (V(L)) and four constant (C(L)) isotypes. The C(L) regions contained three conserved cysteine residues that may participate in intra- or inter-chain disulfide bond formation. The four C(L) isotypes could be sub-grouped into two serological types: κ (C(L)-I, C(L)-II and C(L)-III) and σ (C(L)-IV), by phylogenetic analysis. The OsgIgL genes were found to be expressed in various tissues, with greatest levels of expression observed in the head-kidney and spleen. The major expression type was C(L)-I, which comprised 92% and 91% of total OsgIgL gene expression in the head-kidney and spleen, respectively. Transcription of all four C(L) isotypes was differentially affected in response to various immunostimulators, including lipopolysaccharide (LPS), poly I:C and grouper iridovirus (GIV). Induction of OsgIgL genes in response to immunostimulators was particularly dramatic in the spleen, suggesting this organ holds particular importance for the regulation of OsgIgL expression. Furthermore, vaccination of grouper with formalin-inactivated GIV also induced differential patterns of expression in all four OsgIgL isotypes. In summary, the significant and diverse patterns of transcriptional induction observed for OsgIgL isotypes in the spleen and head-kidney imply that each isotype may have unique roles in the immune response.

  11. Abnormal FISH in patients with immunoglobulin light chain amyloidosis is a risk factor for cardiac involvement and for death

    PubMed Central

    Warsame, R; Kumar, S K; Gertz, M A; Lacy, M Q; Buadi, F K; Hayman, S R; Leung, N; Dingli, D; Lust, J A; Ketterling, R P; Lin, Y; Russell, S; Hwa, L; Kapoor, P; Go, R S; Zeldenrust, S R; Kyle, R A; Rajkumar, S V; Dispenzieri, A

    2015-01-01

    Importance of interphase fluorescent in situ hybridization (FISH) with cytoplasmic staining of immunoglobulin FISH (cIg-FISH) on bone marrow is not well understood in light chain amyloidosis (AL). This is in contrast with multiple myeloma where prognostic and treatment related decisions are dependent on cytogenetic testing. This retrospective study reviewed 401 AL patients with cIg-FISH testing performed at our institution between 2004 and 2012. Eighty-one percent of patients had an abnormal cIg-FISH. Common abnormalities involved translocations of chromosome 14q32 (52%), specifically: t(11;14) (43%), t(14;16) (3%) and t(4;14) (2%). Other common abnormalities include monosomy 13/deletion 13q (30%), trisomies 9 (20%), 15 (14%), 11 (10%) and 3 (10%). Median overall survival for this cohort of patients is 3.5 years. When plasma cell burden was greater than 10% trisomies predicted for worse survival (44 vs 19 months), and when it was ⩽10% t(11;14) predicted for worse survival (53 months vs not reached). Abnormal cIg-FISH was significantly associated with advanced cardiac involvement, and remained a prognostic factor on multivariate analysis. This large AL cohort demonstrates that abnormal FISH at diagnosis is prognostic for survival and advanced cardiac disease. Particularly, trisomies and t(11;14) affect survival when degree of plasma cell burden is considered. PMID:25933374

  12. High prevalence of immunoglobulin light chain gene aberrations as revealed by FISH in multiple myeloma and MGUS.

    PubMed

    Türkmen, Seval; Binder, Anastasia; Gerlach, Antje; Niehage, Sylke; Theodora Melissari, Maria; Inandiklioglu, Nihal; Dörken, Bernd; Burmeister, Thomas

    2014-08-01

    Multiple myeloma (MM) is a malignant B-cell neoplasm characterized by an uncontrolled proliferation of aberrant plasma cells in the bone marrow. Chromosome aberrations in MM are complex and represent a hallmark of the disease, involving many chromosomes that are altered both numerically and structurally. Nearly half of the cases are nonhyperdiploid and show IGH translocations with the following partner genes: CCND1, FGFR3 and MMSET, MAF, MAFB, and CCND3. The remaining 50% are grouped into a hyperdiploid group that is characterized by multiple trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. In this study, we analyzed the immunoglobulin light chain kappa (IGK, 2p12) and lambda (IGL, 22q11) loci in 150 cases, mostly with MM but in a few cases monoclonal gammopathy of undetermined significance (MGUS), without IGH translocations. We identified aberrations in 27% (= 40 patients) including rearrangements (12%), gains (12%), and deletions (4.6%). In 6 of 18 patients with IGK or/and IGL rearrangements, we detected a MYC rearrangement which suggests that MYC is the translocation partner in the majority of these cases. PMID:24729354

  13. Generation of immunoglobulin light chain gene diversity in Raja erinacea is not associated with somatic rearrangement, an exception to a central paradigm of B cell immunity

    PubMed Central

    1995-01-01

    In all vertebrate species examined to date, rearrangement and somatic modification of gene segmental elements that encode portions of the antigen-combining sites of immunoglobulins are integral components of the generation of antibody diversity. In the phylogenetically primitive cartilaginous fishes, gene segments encoding immunoglobulin heavy and light chain loci are arranged in multiple clusters, in which segmental elements are separated by only 300-400 bp. In some cases, segmental elements are joined in the germline of nonlymphoid cells (joined genes). Both genomic library screening and direct amplification of genomic DNA have been used to characterize at least 89 different type I light chain gene clusters in the skate, Raja. Analyses of predicted nucleotide sequences and predicted peptide structures are consistent with the distribution of genes into different sequence groups. Predicted amino acid sequence differences are preferentially distributed in complementarity-determining versus framework regions, and replacement-type substitutions exceed neutral substitutions. When specific germline sequences are related to the sequences of individual cDNAs, it is apparent that the joined genes are expressed and are potentially somatically mutated. No evidence was found for the presence of any type I light chain gene in Raja that is not germline joined. The type I light chain gene clusters in Raja appear to represent a novel gene system in which combinatorial and junctional diversity are absent. PMID:7790811

  14. Immunoglobulin Free Light Chains and GAGs Mediate Multiple Myeloma Extracellular Vesicles Uptake and Secondary NfκB Nuclear Translocation

    PubMed Central

    Di Noto, Giuseppe; Chiarini, Marco; Paolini, Lucia; Mazzoldi, Elena Laura; Giustini, Viviana; Radeghieri, Annalisa; Caimi, Luigi; Ricotta, Doris

    2014-01-01

    Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. Monoclonal plasma cells often secrete high amounts of immunoglobulin free light chains (FLCs) that could induce tissue damage. Recently, we showed that FLCs are internalized in endothelial and myocardial cell lines and secreted in extracellular vesicles (EVs). MM serum derived EVs presented phenotypic differences if compared with monoclonal gammopathy of undetermined significance (MGUS) serum derived EVs suggesting their involvement in MM pathogenesis or progression. To investigate the effect of circulating EVs on endothelial and myocardial cells, we purified MM and MGUS serum derived EVs with differential ultracentrifugation protocols and tested their biological activity. We found that MM and MGUS EVs induced different proliferation and internalization rates in endothelial and myocardial cells, thus we tried to find specific targets in MM EVs docking and processing. Pre-treatment of EVs with anti-FLCs antibodies or heparin blocked the MM EVs uptake, highlighting that FLCs and glycosaminoglycans are involved. Indeed, only MM EVs exposure induced a strong nuclear factor kappa B nuclear translocation that was completely abolished after anti-FLCs antibodies and heparin pre-treatment. The protein tyrosine kinase c-src is present on MM circulating EVs and redistributes to the cell plasma membrane after MM EVs exposure. The anti-FLCs antibodies and heparin pre-treatments were able to block the intracellular re-distribution of the c-src kinase and the subsequent c-src kinase containing EVs production. Our results open new insights in EVs cellular biology and in MM therapeutic and diagnostic approaches. PMID:25386176

  15. Immunoglobulin heavy/light chain ratios improve paraprotein detection and monitoring, identify residual disease and correlate with survival in multiple myeloma patients

    PubMed Central

    Ludwig, H; Milosavljevic, D; Zojer, N; Faint, J M; Bradwell, A R; Hübl, W; Harding, S J

    2013-01-01

    The novel heavy/light chain (HLC) assay was used for the detection and measurement of monoclonal immunoglobulins, response evaluation and prognostication. This test allows identification and quantification of the different light chain types of each immunoglobulin class (for example, IgGκ and IgGλ) and enables calculation of ratios of monoclonal/polyclonal immunoglobulin (HLC ratio). Sequential sera of 156 patients with IgG or IgA myeloma started on first-line therapy and followed for a median of 46.1 months were analyzed. Results were compared with those obtained with conventional techniques (serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE), nephelometry (NEPH), and the free light chain test (FLC)). Our data show that the HLC assay allowed quantification of monoclonal proteins not accurately measurable by SPEP or NEPH. When both HLC and FLC testing were applied for response assessment, clonal excess was noted in 14/31 patients with complete response (CR). HLC ratio indicated presence of disease in 8/31 patients who achieved CR and, in sequential studies indicated evolving relapse in three patients before IFE became positive. Highly abnormal HLC ratios at presentation were significantly associated with shorter overall survival (40.5 months vs median not reached, P=0.016). Multivariate analysis revealed HLC ratio (P=0.03) and β2-microglobulin (P<0.01) as independent risk factors for survival. PMID:22955329

  16. Surrogate or conventional light chains are required for membrane immunoglobulin mu to activate the precursor B cell transition [published erratum appears in J Exp Med 1997 Jan 6;185(1):183

    PubMed Central

    1996-01-01

    To examine the role of light chains in early B cell development we combined RAG-1 and lambda 5 mutations to produce mice that expressed neither conventional nor surrogate light chains (RAG-1-/-, lambda 5-/- ). Unique heavy and light chain genes were then introduced into the double and single mutant backgrounds. Membrane immunoglobulin (Ig)mu (mIg mu) associated with Ig alpha-Ig beta but was unable to activate the pre-B cell transition in RAG-1-/-lambda 5-/- mice. Either lambda 5 or kappa light chains were sufficient to complement this deficiency. Therefore light chains are absolutely required for a functional Ig signaling module in early B cell development. Our data provide direct evidence for the existence of two pathways for induction of early B cell development: one which is activated through surrogate light chains and mIg mu, and an alternative pathway which uses conventional light chains and mIg mu. PMID:8920890

  17. Expression of v-rel induces mature B-cell lines that reflect the diversity of avian immunoglobulin heavy- and light-chain rearrangements.

    PubMed Central

    Barth, C F; Humphries, E H

    1988-01-01

    The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte. Images PMID:2854197

  18. [Secondary monoclonal gammopathy after bone marrow autotransplantation as a cause of worse renal function in light chain immunoglobulin deposition disease].

    PubMed

    Rekhtina, I G; Mendeleeva, L P; Stolyarevich, E S; Gal'tseva, I V; Povilaitite, P E; Biryukova, L S

    2016-01-01

    The paper describes a clinical case of a female woman with nephropathy due to light chain deposition disease caused by secretion of κ Bence-Jones protein. Complete immunochemical remission was achieved after induction therapy using a bortezomib + cyclophosphamide + dexamethasone regimen. Renal function remained unchanged (glomerular filtration rate 16 ml/min), there was a reduction in proteinuria from 5.8 to 2.6 g/day. High-dose melphalan (200 mg/m2) chemotherapy with peripheral blood stem cell autotransplantation was performed as consolidation of remission. A year posttransplantation, there was no secretion of κ light chains; however, monoclonal IgG lambda emerged in a quantity of 3.2 g/l. At the same period, nephrotic syndrome became progressive (daily proteinuria 12 g) and dialysis-dependent renal failure developed. A repeat renal biopsy specimen revealed changes, suggesting that there was a decrease in renal deposits of κ light chains. Simultaneously with this, the obvious negative trend as progressive nephrosclerosis and fixation of IgG and λ light chains in the glomeruli (in the sclerotic areas) cause IgGλ monoclonal protein to be involved in the genesis of further kidney injury. Attention is also paid to different characteristics of capillary wall deposits by density (according to the electron microscopic findings), which may point to their different qualitative composition and possibly different formation duration. Papaprotein Gλ disappeared after a year without therapy, suggesting its reactivity. The findings confirm that worse renal function is caused by the action of paraprotein Gλ due to secondary (after autologous hematopoietic stem cells transplantation) monoclonal gammopathy.

  19. [Secondary monoclonal gammopathy after bone marrow autotransplantation as a cause of worse renal function in light chain immunoglobulin deposition disease].

    PubMed

    Rekhtina, I G; Mendeleeva, L P; Stolyarevich, E S; Gal'tseva, I V; Povilaitite, P E; Biryukova, L S

    2016-01-01

    The paper describes a clinical case of a female woman with nephropathy due to light chain deposition disease caused by secretion of κ Bence-Jones protein. Complete immunochemical remission was achieved after induction therapy using a bortezomib + cyclophosphamide + dexamethasone regimen. Renal function remained unchanged (glomerular filtration rate 16 ml/min), there was a reduction in proteinuria from 5.8 to 2.6 g/day. High-dose melphalan (200 mg/m2) chemotherapy with peripheral blood stem cell autotransplantation was performed as consolidation of remission. A year posttransplantation, there was no secretion of κ light chains; however, monoclonal IgG lambda emerged in a quantity of 3.2 g/l. At the same period, nephrotic syndrome became progressive (daily proteinuria 12 g) and dialysis-dependent renal failure developed. A repeat renal biopsy specimen revealed changes, suggesting that there was a decrease in renal deposits of κ light chains. Simultaneously with this, the obvious negative trend as progressive nephrosclerosis and fixation of IgG and λ light chains in the glomeruli (in the sclerotic areas) cause IgGλ monoclonal protein to be involved in the genesis of further kidney injury. Attention is also paid to different characteristics of capillary wall deposits by density (according to the electron microscopic findings), which may point to their different qualitative composition and possibly different formation duration. Papaprotein Gλ disappeared after a year without therapy, suggesting its reactivity. The findings confirm that worse renal function is caused by the action of paraprotein Gλ due to secondary (after autologous hematopoietic stem cells transplantation) monoclonal gammopathy. PMID:27296267

  20. Compensatory Aspects of Allele Diversity at Immunoglobulin Loci: Gene Correlations in Rabbit Populations Devoid of Light Chain Diversity (Oryctolagus Cuniculus L.; Kerguelen Islands)

    PubMed Central

    van-der-Loo, W.; Bousses, P.; Arthur, C. P.; Chapuis, J. L.

    1996-01-01

    Is there a selective advantage of increased diversity at one immunoglobulin locus when diversity at another locus is low? A previous paper demonstrated excess heterozygosity at the rabbit light chain b locus when heterozygosity was low at the heavy chain constant region e locus. Here we consider the reverse situation by analyzing allele distributions at heavy chain loci in populations fixed for the light chain b locus. We analyzed the a locus that encodes the predominantly expressed heavy chain variable region, and the d and e loci that control different parts of the Ig gamma class constant region. While there was excess heterozygosity, genetic differentiation between localities was extensive and was most pronounced for females. This was in marked contrast with observations in areas where b-locus diversity was important and confirms a negative correlation between e- and b-locus heterozygosity. Trigenic disequilibria corresponded to a significant negative correlation between e- and a-locus heterozygosity due mainly to strong variation among localities within the context of pronounced (digenic) linkage disequilibria. Although substantial, the average increase in a/e-locus single heterozygosity implemented by higher order disequilibria within localities was not significant. PMID:8913759

  1. Immunoglobulin-free light chain monomer-dimer patterns help to distinguish malignant from premalignant monoclonal gammopathies: a pilot study.

    PubMed

    Kaplan, Batia; Golderman, Sizilia; Aizenbud, Boris; Esev, Konstantin; Kukuy, Olga; Leiba, Merav; Livneh, Avi; Ben-Zvi, Ilan

    2014-09-01

    Multiple myeloma (MM) and AL amyloidosis (AL) are two malignant forms of monoclonal gammopathies. For the purposes of prognosis and treatment, it is important to distinguish these diseases from the premalignant forms of monoclonal gammopathies, such as monoclonal gammopathy of unknown significance (MGUS) and smoldering myeloma (SMM). Routine serum/urine tests for monoclonal protein are insufficient for differential diagnosis. Thus, invasive procedures, such as tissue aspiration or biopsy, are applied. In this study, we aimed at characterization of serum-free light chain (FLC) monomer-dimer patterns to distinguish the malignant from the premalignant forms of monoclonal gammopathies. A quantitative Western blotting was applied to estimate the FLC monomer and dimer levels in AL, MM, MGUS, and SMM patients, and in control subjects (healthy individuals and patients with AA amyloidosis). AL and MM patients displayed an abnormally increased dimerization of monoclonal FLC, accompanied by higher clonality values of FLC dimers, as compared to that of monomers. These abnormalities of FLC patterns were not observed in patients with MGUS, SMM, AA amyloidosis, and healthy individuals. Analysis of FLC patterns helped to differentiate AL and MM from MGUS and SMM, a goal difficult to achieve using routine serum tests. Also, our technique might serve as a complimentary diagnostic tool in the cases with suspected AL amyloidosis, where the diagnosis of MM is excluded, while the results of amyloid typing by routine immunohistochemical techniques are inconclusive. PMID:24866208

  2. The flow cytometry-defined light chain cytoplasmic immunoglobulin index and an associated 12-gene expression signature are independent prognostic factors in multiple myeloma.

    PubMed

    Papanikolaou, X; Alapat, D; Rosenthal, A; Stein, C; Epstein, J; Owens, R; Yaccoby, S; Johnson, S; Bailey, C; Heuck, C; Tian, E; Joiner, A; van Rhee, F; Khan, R; Zangari, M; Jethava, Y; Waheed, S; Davies, F; Morgan, G; Barlogie, B

    2015-08-01

    As part of Total Therapy (TT) 3b, baseline marrow aspirates were subjected to two-color flow cytometry of nuclear DNA content and cytoplasmic immunoglobulin (DNA/CIG) as well as plasma cell gene expression profiling (GEP). DNA/CIG-derived parameters, GEP and standard clinical variables were examined for their effects on overall survival (OS) and progression-free survival (PFS). Among DNA/CIG parameters, the percentage of the light chain-restricted (LCR) cells and their cytoplasmic immunoglobulin index (CIg) were linked to poor outcome. In the absence of GEP data, low CIg <2.8, albumin <3.5 g/dl and age ⩾65 years were significantly associated with inferior OS and PFS. When GEP information was included, low CIg survived the model along with GEP70-defined high risk and low albumin. Low CIg was linked to beta-2-microglobulin >5.5 mg/l, a percentage of LCR cells exceeding 50%, C-reactive protein ⩾8 mg/l and GEP-derived high centrosome index. Further analysis revealed an association of low CIg with 12 gene probes implicated in cell cycle regulation, differentiation and drug transportation from which a risk score was developed in TT3b that held prognostic significance also in TT3a, TT2 and HOVON trials, thus validating its general applicability. Low CIg is a powerful new prognostic variable and has identified potentially drug-able targets. PMID:25753926

  3. Somatic mutation and CDR3 lengths of immunoglobulin kappa light chains expressed in patients with rheumatoid arthritis and in normal individuals.

    PubMed Central

    Bridges, S L; Lee, S K; Johnson, M L; Lavelle, J C; Fowler, P G; Koopman, W J; Schroeder, H W

    1995-01-01

    Immunoglobulin secretion by plasma cells infiltrating synovial membranes is a prominent feature of RA. Previous analyses of a cDNA library generated from synovium of RA patient BC revealed immunoglobulin kappa light chain transcripts with extensive somatic mutation, frequent N region addition, and unexpected variation in the lengths of CDR3 regions which form the center of the antigen binding site. To determine if these characteristics are present in other individuals, we performed reverse transcription-polymerase chain reaction amplification and sequenced > or = 10 V kappa-containing amplicons from nine tissue samples: synovia of three individuals with long-standing RA (including patient BC), PBLs of two of these individuals, and PBLs or splenocytes of four normal individuals. Increased levels of somatic mutation in PBLs appeared to correlate with increased age, which may reflect accumulation of circulating memory cells and/or decreased bone marrow production of naive B lymphocytes. Two of three RA synovial samples and both RA PBL samples exhibited increased proportions of clones with unusual CDR3 lengths. Enrichment for these antibody binding sites could be due to abnormal regulation of the emerging repertoire or to selection for B lymphocytes bearing antibodies of unusual specificity, and may play a role in the pathogenesis of RA. Images PMID:7635977

  4. The flow cytometry-defined light chain cytoplasmic immunoglobulin index and an associated 12-gene expression signature are independent prognostic factors in multiple myeloma.

    PubMed

    Papanikolaou, X; Alapat, D; Rosenthal, A; Stein, C; Epstein, J; Owens, R; Yaccoby, S; Johnson, S; Bailey, C; Heuck, C; Tian, E; Joiner, A; van Rhee, F; Khan, R; Zangari, M; Jethava, Y; Waheed, S; Davies, F; Morgan, G; Barlogie, B

    2015-08-01

    As part of Total Therapy (TT) 3b, baseline marrow aspirates were subjected to two-color flow cytometry of nuclear DNA content and cytoplasmic immunoglobulin (DNA/CIG) as well as plasma cell gene expression profiling (GEP). DNA/CIG-derived parameters, GEP and standard clinical variables were examined for their effects on overall survival (OS) and progression-free survival (PFS). Among DNA/CIG parameters, the percentage of the light chain-restricted (LCR) cells and their cytoplasmic immunoglobulin index (CIg) were linked to poor outcome. In the absence of GEP data, low CIg <2.8, albumin <3.5 g/dl and age ⩾65 years were significantly associated with inferior OS and PFS. When GEP information was included, low CIg survived the model along with GEP70-defined high risk and low albumin. Low CIg was linked to beta-2-microglobulin >5.5 mg/l, a percentage of LCR cells exceeding 50%, C-reactive protein ⩾8 mg/l and GEP-derived high centrosome index. Further analysis revealed an association of low CIg with 12 gene probes implicated in cell cycle regulation, differentiation and drug transportation from which a risk score was developed in TT3b that held prognostic significance also in TT3a, TT2 and HOVON trials, thus validating its general applicability. Low CIg is a powerful new prognostic variable and has identified potentially drug-able targets.

  5. Heavy-light chain interrelations of MS-associated immunoglobulins probed by deep sequencing and rational variation.

    PubMed

    Lomakin, Yakov A; Zakharova, Maria Yu; Stepanov, Alexey V; Dronina, Maria A; Smirnov, Ivan V; Bobik, Tatyana V; Pyrkov, Andrey Yu; Tikunova, Nina V; Sharanova, Svetlana N; Boitsov, Vitali M; Vyazmin, Sergey Yu; Kabilov, Marsel R; Tupikin, Alexey E; Krasnov, Alexey N; Bykova, Nadezda A; Medvedeva, Yulia A; Fridman, Marina V; Favorov, Alexander V; Ponomarenko, Natalia A; Dubina, Michael V; Boyko, Alexey N; Vlassov, Valentin V; Belogurov, Alexey A; Gabibov, Alexander G

    2014-12-01

    The mechanisms triggering most of autoimmune diseases are still obscure. Autoreactive B cells play a crucial role in the development of such pathologies and, in particular, production of autoantibodies of different specificities. The combination of deep-sequencing technology with functional studies of antibodies selected from highly representative immunoglobulin combinatorial libraries may provide unique information on specific features in the repertoires of autoreactive B cells. Here, we have analyzed cross-combinations of the variable regions of human immunoglobulins against the myelin basic protein (MBP) previously selected from a multiple sclerosis (MS)-related scFv phage-display library. On the other hand, we have performed deep sequencing of the sublibraries of scFvs against MBP, Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and myelin oligodendrocyte glycoprotein (MOG). Bioinformatics analysis of sequencing data and surface plasmon resonance (SPR) studies have shown that it is the variable fragments of antibody heavy chains that mainly determine both the affinity of antibodies to the parent autoantigen and their cross-reactivity. It is suggested that LMP1-cross-reactive anti-myelin autoantibodies contain heavy chains encoded by certain germline gene segments, which may be a hallmark of the EBV-specific B cell subpopulation involved in MS triggering.

  6. A Comprehensive Analysis of the Phylogeny, Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis, an Endangered Reptile Species.

    PubMed

    Wang, Xifeng; Cheng, Gang; Lu, Yan; Zhang, Chenglin; Wu, Xiaobing; Han, Haitang; Zhao, Yaofeng; Ren, Liming

    2016-01-01

    Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.

  7. A Comprehensive Analysis of the Phylogeny, Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis, an Endangered Reptile Species

    PubMed Central

    Lu, Yan; Zhang, Chenglin; Wu, Xiaobing; Han, Haitang; Zhao, Yaofeng; Ren, Liming

    2016-01-01

    Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates. PMID:26901135

  8. A Comprehensive Analysis of the Phylogeny, Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis, an Endangered Reptile Species.

    PubMed

    Wang, Xifeng; Cheng, Gang; Lu, Yan; Zhang, Chenglin; Wu, Xiaobing; Han, Haitang; Zhao, Yaofeng; Ren, Liming

    2016-01-01

    Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates. PMID:26901135

  9. Diagnostic and Prognostic Value of the Cerebrospinal Fluid Concentration of Immunoglobulin Free Light Chains in Clinically Isolated Syndrome with Conversion to Multiple Sclerosis

    PubMed Central

    Makshakov, Gleb; Nazarov, Vladimir; Kochetova, Olga; Surkova, Elena; Lapin, Sergey; Evdoshenko, Evgeniy

    2015-01-01

    Background and objective In this study, we evaluated the diagnostic and prognostic significance of cerebrospinal fluid free light chains (CSF FLC) at the time of clinically isolated syndrome (CIS). Methods We compared FLC-parameters at the moment of CIS in patients with conversion to multiple sclerosis (MS) after 2 years (CIS-MS), patients who remained stable both clinically and radiologically after 2 years (CIS-nonMS), patients with non-inflammatory neurologic diseases (NIND) as a comparison group and patients with other inflammatory neurologic diseases (IND) with intrathecal oligoclonal bands (OCB) synthesis. ROC-analysis was conducted to define FLC-assay characteristics and cut-off values. We also compared FLC-concentrations in CIS patients to determine their OCB-status. A correlation analysis was performed between FLC-concentrations and the expanded disability scale score (EDSS), annualized relapse rate (ARR) and MRI-activity (i.e., number of new and gadolinium-enhancing (Gd+) lesions) in patients. Results The levels of kappa-FLC (k-FLCCSF) and lambda-FLC (λ-FLCCSF) as well as kappa- and lambda-quotients (Q-k and Q-λ) were elevated in CIS-MS compared to the CIS-nonMS and NIND groups. These levels did not differ significantly when compared with the IND group. We identified several patients with high k-FLCCSF and λ-FLCCSF in OCB-negative CIS and IND groups. The level of k-FLCCSF production was significantly higher in OCB-positive patients in the CIS-MS group compared to the CIS-nonMS group. The concentrations of k-FLCCSF and Q-k in the CIS-MS group showed significant correlation with the level of EDSS after 2 years (k-FLC: r = 0.4477,p = 0.0016; Q-k: r = 0.4621, p = 0.0016). λ-FLCCSF and Q-λ inversely correlated with the number of Gd+ lesions (CSF λ-FLC: r = -0.3698, p = 0.0223; Q-λ: r = -0.4527, p = 0.0056). Conclusion The concentration of CSF FLC predicts conversion to MS within 2 years following CIS. OCB-positive patients with an early conversion have a

  10. Human placenta: relative content of antibodies of different classes and subclasses (IgG1-IgG4) containing lambda- and kappa-light chains and chimeric lambda-kappa-immunoglobulins.

    PubMed

    Lekchnov, Evgenii A; Sedykh, Sergey E; Dmitrenok, Pavel S; Buneva, Valentina N; Nevinsky, Georgy A

    2015-06-01

    The specific organ placenta is much more than a filter: it is an organ that protects, feeds and regulates the growth of the embryo. Affinity chromatography, ELISA, SDS-PAGE and matrix-assisted laser desorption ionization mass spectrometry were used. Using 10 intact human placentas deprived of blood, a quantitative analysis of average relative content [% of total immunoglobulins (Igs)] was carried out for the first time: (92.7), IgA (2.4), IgM (2.5), kappa-antibodies (51.4), lambda-antibodies (48.6), IgG1 (47.0), IgG2 (39.5), IgG3 (8.8) and IgG4 (4.3). It was shown for the first time that placenta contains sIgA (2.5%). In the classic paradigm, Igs represent products of clonal B-cell populations, each producing antibodies recognizing a single antigen. There is a common belief that IgGs in mammalian biological fluids are monovalent molecules having stable structures and two identical antigen-binding sites. However, similarly to human milk Igs, placenta antibodies undergo extensive half-molecule exchange and the IgG pool consists of 43.5 ± 15.0% kappa-kappa-IgGs and 41.6 ± 17.0% lambda-lambda-IgGs, while 15.0 ± 4.0% of the IgGs contained both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained, respectively (%): IgG1 (47.7 and 34.4), IgG2 (36.3 and 44.5), IgG3 (7.4 and 11.8) and IgG4 (7.5 and 9.1), while chimeric kappa-lambda-IgGs consisted of (%): 43.5 IgG1, 41.0 IgG2, 5.6 IgG3 and 7.9 IgG4. Our data are indicative of the possibility of half-molecule exchange between placenta IgGs of various subclasses, raised against different antigens, which explains a very well-known polyspecificity and cross-reactivity of different human IgGs.

  11. Serum free light chain assays not total light chain assays are the standard of care to assess Monoclonal Gammopathies.

    PubMed

    Tietsche de Moraes Hungria, Vania; Allen, Syreeta; Kampanis, Petros; Soares, Elyara Maria

    2016-01-01

    The diagnosis of Multiple Myeloma is a challenge to the physician due to the non-specific symptoms (anemia, bone pain and recurrent infections) that are commonplace in the elderly population. However, early diagnosis is associated with less severe disease, including fewer patients presenting with acute renal injury, pathological fractures and severe anemia. Since 2006, the serum free light chain test Freelite(®) has been included alongside standard laboratory tests (serum and urine protein electrophoresis, and serum and urine immunofixation) as an aid in the identification of monoclonal proteins, which are a cornerstone for the diagnosis of Multiple Myeloma. The serum free light chain assay recognizes the light chain component of the immunoglobulin in its free form with high sensitivity. Other assays that measure light chains in the free and intact immunoglobulin forms are sensitive, but unfortunately, due to the nomenclature used, these assays (total light chains) are sometimes used in place of the free light chain assay. This paper reviews the available literature comparing the two assays and tries to clarify hypothetical limitations of the total assay to detect Multiple Myeloma. Furthermore, we elaborate on our study comparing the two assays used in 11 Light Chain Multiple Myeloma patients at presentation and 103 patients taken through the course of their disease. The aim of this article is to provide a clear discrimination between the two assays and to provide information to physicians and laboratory technicians so that they can utilize the International Myeloma Working Group guidelines.

  12. Serum free light chain assays not total light chain assays are the standard of care to assess Monoclonal Gammopathies

    PubMed Central

    Tietsche de Moraes Hungria, Vania; Allen, Syreeta; Kampanis, Petros; Soares, Elyara Maria

    2016-01-01

    The diagnosis of Multiple Myeloma is a challenge to the physician due to the non-specific symptoms (anemia, bone pain and recurrent infections) that are commonplace in the elderly population. However, early diagnosis is associated with less severe disease, including fewer patients presenting with acute renal injury, pathological fractures and severe anemia. Since 2006, the serum free light chain test Freelite® has been included alongside standard laboratory tests (serum and urine protein electrophoresis, and serum and urine immunofixation) as an aid in the identification of monoclonal proteins, which are a cornerstone for the diagnosis of Multiple Myeloma. The serum free light chain assay recognizes the light chain component of the immunoglobulin in its free form with high sensitivity. Other assays that measure light chains in the free and intact immunoglobulin forms are sensitive, but unfortunately, due to the nomenclature used, these assays (total light chains) are sometimes used in place of the free light chain assay. This paper reviews the available literature comparing the two assays and tries to clarify hypothetical limitations of the total assay to detect Multiple Myeloma. Furthermore, we elaborate on our study comparing the two assays used in 11 Light Chain Multiple Myeloma patients at presentation and 103 patients taken through the course of their disease. The aim of this article is to provide a clear discrimination between the two assays and to provide information to physicians and laboratory technicians so that they can utilize the International Myeloma Working Group guidelines. PMID:26969773

  13. Neurofilament light chain

    PubMed Central

    Lu, Ching-Hua; Macdonald-Wallis, Corrie; Gray, Elizabeth; Pearce, Neil; Petzold, Axel; Norgren, Niklas; Giovannoni, Gavin; Fratta, Pietro; Sidle, Katie; Fish, Mark; Orrell, Richard; Howard, Robin; Talbot, Kevin; Greensmith, Linda; Kuhle, Jens

    2015-01-01

    Objective: To test blood and CSF neurofilament light chain (NfL) levels in relation to disease progression and survival in amyotrophic lateral sclerosis (ALS). Methods: Using an electrochemiluminescence immunoassay, NfL levels were measured in samples from 2 cohorts of patients with sporadic ALS and healthy controls, recruited in London (ALS/control, plasma: n = 103/42) and Oxford (ALS/control, serum: n = 64/36; paired CSF: n = 38/20). NfL levels in patients were measured at regular intervals for up to 3 years. Change in ALS Functional Rating Scale–Revised score was used to assess disease progression. Survival was evaluated using Cox regression and Kaplan–Meier analysis. Results: CSF, serum, and plasma NfL discriminated patients with ALS from healthy controls with high sensitivity (97%, 89%, 90%, respectively) and specificity (95%, 75%, 71%, respectively). CSF NfL was highly correlated with serum levels (r = 0.78, p < 0.0001). Blood NfL levels were approximately 4 times as high in patients with ALS compared with controls in both cohorts, and maintained a relatively constant expression during follow-up. Blood NfL levels at recruitment were strong, independent predictors of survival. The highest tertile of blood NfL at baseline had a mortality hazard ratio of 3.91 (95% confidence interval 1.98–7.94, p < 0.001). Conclusion: Blood-derived NfL level is an easily accessible biomarker with prognostic value in ALS. The individually relatively stable levels longitudinally offer potential for NfL as a pharmacodynamic biomarker in future therapeutic trials. Classification of evidence: This report provides Class III evidence that the NfL electrochemiluminescence immunoassay accurately distinguishes patients with sporadic ALS from healthy controls. PMID:25934855

  14. Light-chain binding sites on renal brush-border membranes

    SciTech Connect

    Batuman, V.; Dreisbach, A.W.; Cyran, J.

    1990-05-01

    Immunoglobulin light chains are low-molecular-weight proteins filtered at the renal glomerulus and catabolized within the proximal tubular epithelium. Excessive production and urinary excretion of light chains are associated with renal dysfunction. They also interfere with proximal renal tubule epithelial functions in vitro. We studied the binding of 125I-labeled kappa- and lambda-light chains, obtained from the urine of multiple myeloma patients, to rat and human renal proximal tubular brush-border membranes. Light-chain binding to brush borders was also demonstrated immunologically by flow cytometry. Computer analysis of binding data was consistent with presence of a single class of low-affinity, high-capacity, non-cooperative binding sites with relative selectivity for light chains on both rat and human kidney brush-border membranes. The dissociation constants of light chains ranged from 1.6 X 10(-5) to 1.2 X 10(-4) M, and maximum binding capacity ranged from 4.7 +/- 1.3 X 10(-8) to 8.0 +/- 0.9 X 10(-8) (SD) mol/mg protein at 25 degrees C. Kappa- and lambda-light chains competed with each other for binding with comparable affinity constants. Competition by albumin and beta-lactoglobulin, however, was much weaker, suggesting relative site selectivity for light chains. These binding sites probably function as endocytotic receptors for light chains and possibly other low-molecular-weight proteins.

  15. The morphologic spectrum and clinical significance of light chain proximal tubulopathy with and without crystal formation.

    PubMed

    Larsen, Christopher P; Bell, Jane M; Harris, Alexis A; Messias, Nidia C; Wang, Yihan H; Walker, Patrick D

    2011-11-01

    The renal diseases most frequently associated with myeloma include amyloidosis, monoclonal immunoglobulin deposition disease, and cast nephropathy. Less frequently reported is light chain proximal tubulopathy, a disease characterized by κ-restricted crystal deposits in the proximal tubule cytoplasm. Light chain proximal tubulopathy without crystal deposition is only loosely related to the typical light chain proximal tubulopathy, and little is known about this entity. A search was performed of the 10 081 native kidney biopsy samples processed by our laboratory over the past 2 years for cases that had light chain restriction limited to the proximal tubule cytoplasm. A total of 10 cases of light chain proximal tubulopathy without crystal deposition were found representing 3.1% of light chain-related diseases. Nine of these 10 showed λ-light chain restriction. Only three cases of light chain proximal tubulopathy with crystals were found accounting for 0.9% of light chain-related diseases. Two of these three were κ subtype. Plasma cell dyscrasia was unsuspected in seven of the 10 patients with light chain proximal tubulopathy without crystals at the time of renal biopsy. After the biopsy was reported, follow-up was available on 9/10 patients with 9/9 showing a plasma cell dyscrasia including 8/9 with multiple myeloma. We found that light chain proximal tubulopathy without crystal formation, despite being rarely described in the literature, is over three times more common than light chain proximal tubulopathy with crystal formation in our series. And given that it is often associated with previously unrecognized myeloma, it is a critically important diagnosis.

  16. Serum-free light-chain assay: clinical utility and limitations.

    PubMed

    Bhole, Malini V; Sadler, Ross; Ramasamy, Karthik

    2014-09-01

    In the last decade, the introduction of the serum-free light-chain (sFLC) assay has been an important advance in the diagnosis and management of plasma cell dyscrasias, particularly monoclonal light-chain diseases. The immunoassay was developed to detect free light chains in serum by using anti-FLC antibodies which specifically recognised epitopes on light chains that were 'hidden' in intact immunoglobulins. Since its introduction in 2001, there have been several publications in the English language literature discussing the clinical utility as well as analytical limitations of the sFLC assay. These studies have highlighted both positive and negative aspects of the assay particularly with regard to its sensitivity and specificity and the technical challenges that can affect its performance. The contribution and significance of the sFLC assay in the management of light-chain myeloma, primary amyloid light-chain (AL) amyloidosis and non-secretory myeloma are well recognised and will be addressed in this review. The aim of this article is to also review the published literature with a view to providing a clear understanding of its utility and limitations in the diagnosis, prognosis and monitoring of plasma dyscrasias including intact immunoglobulin multiple myeloma (MM) and monoclonal gammopathy of unknown significance (MGUS). The increasing interest in using this assay in other haematological conditions will also be briefly discussed. PMID:24489083

  17. Serum-free light-chain assay: clinical utility and limitations.

    PubMed

    Bhole, Malini V; Sadler, Ross; Ramasamy, Karthik

    2014-09-01

    In the last decade, the introduction of the serum-free light-chain (sFLC) assay has been an important advance in the diagnosis and management of plasma cell dyscrasias, particularly monoclonal light-chain diseases. The immunoassay was developed to detect free light chains in serum by using anti-FLC antibodies which specifically recognised epitopes on light chains that were 'hidden' in intact immunoglobulins. Since its introduction in 2001, there have been several publications in the English language literature discussing the clinical utility as well as analytical limitations of the sFLC assay. These studies have highlighted both positive and negative aspects of the assay particularly with regard to its sensitivity and specificity and the technical challenges that can affect its performance. The contribution and significance of the sFLC assay in the management of light-chain myeloma, primary amyloid light-chain (AL) amyloidosis and non-secretory myeloma are well recognised and will be addressed in this review. The aim of this article is to also review the published literature with a view to providing a clear understanding of its utility and limitations in the diagnosis, prognosis and monitoring of plasma dyscrasias including intact immunoglobulin multiple myeloma (MM) and monoclonal gammopathy of unknown significance (MGUS). The increasing interest in using this assay in other haematological conditions will also be briefly discussed.

  18. Cell Damage in Light Chain Amyloidosis: FIBRIL INTERNALIZATION, TOXICITY AND CELL-MEDIATED SEEDING.

    PubMed

    Marin-Argany, Marta; Lin, Yi; Misra, Pinaki; Williams, Angela; Wall, Jonathan S; Howell, Kyle G; Elsbernd, Laura R; McClure, Megan; Ramirez-Alvarado, Marina

    2016-09-16

    Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding, aggregation, and systemic deposition of amyloid composed of immunoglobulin light chains (LC). This work describes our studies on potential mechanisms of AL cytotoxicity. We have studied the internalization of AL soluble proteins and amyloid fibrils into human AC16 cardiomyocytes by using real time live cell image analysis. Our results show how external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation. Soluble protein and external aggregates are internalized into AC16 cells via macropinocytosis. AL amyloid fibrils are shown to be highly cytotoxic at low concentrations. Additionally, caspase assays revealed soluble protein induces apoptosis, demonstrating different cytotoxic mechanisms between soluble protein and amyloid aggregates. This study emphasizes the complex immunoglobulin light chain-cell interactions that result in fibril internalization, protein recruitment, and cytotoxicity that may occur in AL amyloidosis. PMID:27462073

  19. Biased Immunoglobulin Light Chain Gene Usage in the Shark.

    PubMed

    Iacoangeli, Anna; Lui, Anita; Naik, Ushma; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

    2015-10-15

    This study of a large family of κ L chain clusters in nurse shark completes the characterization of its classical Ig gene content (two H chain isotypes, μ and ω, and four L chain isotypes, κ, λ, σ, and σ-2). The shark κ clusters are minigenes consisting of a simple VL-JL-CL array, where V to J recombination occurs over an ~500-bp interval, and functional clusters are widely separated by at least 100 kb. Six out of ~39 κ clusters are prerearranged in the germline (germline joined). Unlike the complex gene organization and multistep assembly process of Ig in mammals, each shark Ig rearrangement, somatic or in the germline, appears to be an independent event localized to the minigene. This study examined the expression of functional, nonproductive, and sterile transcripts of the κ clusters compared with the other three L chain isotypes. κ cluster usage was investigated in young sharks, and a skewed pattern of split gene expression was observed, one similar in functional and nonproductive rearrangements. These results show that the individual activation of the spatially distant κ clusters is nonrandom. Although both split and germline-joined κ genes are expressed, the latter are prominent in young animals and wane with age. We speculate that, in the shark, the differential activation of the multiple isotypes can be advantageously used in receptor editing.

  20. Immunoglobulins and immunoglobulin genes of the horse.

    PubMed

    Wagner, Bettina

    2006-01-01

    Antibodies of the horse were studied intensively by many notable immunologists throughout the past century until the early 1970's. After a large gap of interest in horse immunology, additional basic studies on horse immunoglobulin genes performed during the past 10 years have resulted in new insights into the equine humoral immune system. These include the characterization of the immunoglobulin lambda and kappa light chain genes, the immunoglobulin heavy chain constant (IGHC) gene regions, and initial studies regarding the heavy chain variable genes. Horses express predominately lambda light chains and seem to have a relatively restricted germline repertoire of both lambda and kappa chain variable genes. The IGHC region contains eleven constant heavy chain genes, seven of which are gamma heavy chain genes. It is suggested that all seven genes encoding IgG isotypes are expressed and have distinct functions in equine immune responses.

  1. Equine immunoglobulins and organization of immunoglobulin genes.

    PubMed

    Walther, Stefanie; Rusitzka, Tamara V; Diesterbeck, Ulrike S; Czerny, Claus-Peter

    2015-12-01

    Our understanding of how equine immunoglobulin genes are organized has increased significantly in recent years. For equine heavy chains, 52 IGHV, 40 IGHD, 8 IGHJ and 11 IGHC are present. Seven of these IGHCs are gamma chain genes. Sequence diversity is increasing between fetal, neonatal, foal and adult age. The kappa light chain contains 60 IGKV, 5 IGKJ and 1 IGKC, whereas there are 144 IGLV, 7 IGLJ, and 7 IGLC for the lambda light chain, which is expressed predominantly in horses. Significant transcriptional differences for IGLV and IGLC are identified in different breeds. Allotypic and allelic variants are observed for IGLC1, IGLC5, and IGLC6/7, and two IGLV pseudogenes are also transcribed. During age development, a decrease in IGLVs is noted, although nucleotide diversity and significant differences in gene usage increased. The following paper suggests a standardization of the existing nomenclature of immunoglobulin genes.

  2. A novel antibody light chain dimer: Implications for T-cell receptor structure

    SciTech Connect

    Schiffer, M.; Chang, Chong-Hwan; Solomon, A.; Stevens, F.J.

    1989-01-01

    The dimeric structures of antibody light chains produced in patients with multiple myeloma (Bence Jones proteins) have for some time been studied chemically and crystallographically as models of the antigen binding fragment (Fab) of an antibody. The conformational concordance of Fabs and a Bence Jones dimer was demonstrated by the initial immunoglobulin crystallographic structures. We have recently described the structure of a second intact light chain, the lambda-type protein Loc. The Loc protein exhibits an unanticipated protruding arrangement of its complementarity-determining residues. Grooves on each side of the protrusion may function as separate binding sites. In this report, we examine the Loc structure and its intracrystalline interactions in more detail and consider aspects of this structure that may possess implications for models of a nonantibody constituent of the immunoglobulin superfamily, the T-cell antigen receptor. 26 refs., 3 figs., 1 tab.

  3. Monitoring serum free light chains in patients with multiple myeloma who achieved negative immunofixation after allogeneic stem cell transplantation.

    PubMed

    Mösbauer, Ulrike; Ayuk, Francis; Schieder, Heike; Lioznov, Michael; Zander, Axel R; Kröger, Nicolaus

    2007-02-01

    Monitoring of serum free immunoglobulin light chains (FLC) in 26 myeloma patients who achieved immunofixation negativity after allografting showed a decrease of FLC at a median of 128 days before immunofixation negativity. In patients who subsequently relapsed, a 25% increase of FLC was observed at a median of 98 days before immunofixation positivity.

  4. [Light Chain Amyloidosis: an Update for Treatment].

    PubMed

    Shen, Kai-Ni; Li, Jian

    2015-06-01

    Systemic light chain amyloidosis (AL amyloidosis) is the most common type of amyloidosis, in which deposition of misfolded monoclonal light chain secreted by underlying clonal plasma cells leads to organ dysfunction. Tissue biopsy of involved organ is needed to confirm the type of amyloid deposits, thus proper treatment could be applied. Laser microdissection followed by mass spectrometry, performed on formalin-fixed paraffin-embedded specimens, has been proven superior to traditional methods on accurate diagnosis of amyloidosis. Prognosis depends on the extent of cardiac involvement. The Mayo staging system using NT-ProBNP, cardiac troponin-T and free light chain, is the most robust method for risk stratification and treatment guidance. The introduction of autologous stem cell transplantation (auto-ASCT) resulted in long-term survival in responders, while treatment-related toxicity substantially limited the number of eligible candidates. Novel agents, especially bortezomib, thalidomide and lenalidomide hold promise to achieve comparable hematological responses with auto-ASCT, which might play significant role in treatment of recurrent or refractory AL amyloidosis. PMID:26117060

  5. Natural history and outcome of light chain deposition disease

    PubMed Central

    Sayed, Rabya H.; Wechalekar, Ashutosh D.; Gilbertson, Janet A.; Bass, Paul; Mahmood, Shameem; Sachchithanantham, Sajitha; Fontana, Marianna; Patel, Ketna; Whelan, Carol J.; Lachmann, Helen J.; Hawkins, Philip N.

    2015-01-01

    Light chain deposition disease (LCDD) is characterized by the deposition of monotypic immunoglobulin light chains in the kidney, resulting in renal dysfunction. Fifty-three patients with biopsy-proven LCDD were prospectively followed at the UK National Amyloidosis Center. Median age at diagnosis was 56 years, and patients were followed for a median of 6.2 years (range, 1.1-14.0 years). Median renal survival from diagnosis by Kaplan-Meier analysis was 5.4 years, and median estimated patient survival was 14.0 years; 64% of patients were alive at censor. Sixty-two percent of patients required dialysis, and median survival from commencement of dialysis was 5.2 years. There was a strong association between hematologic response to chemotherapy and renal outcome, with a mean improvement in glomerular filtration rate (GFR) of 6.1 mL/min/year among those achieving a complete or very good partial hematologic response (VGPR) with chemotherapy, most of whom remained dialysis independent, compared with a mean GFR loss of 6.5 mL/min/year among those achieving only a partial or no hematologic response (P < .009), most of whom developed end-stage renal disease (ESRD; P = .005). Seven patients received a renal transplant, and among those whose underlying clonal disorder was in sustained remission, there was no recurrence of LCDD up to 9.7 years later. This study highlights the need to diagnose and treat LCDD early and to target at least a hematologic VGPR with chemotherapy, even among patients with advanced renal dysfunction, to delay progression to ESRD and prevent recurrence of LCDD in the renal allografts of those who subsequently receive a kidney transplant. PMID:26392598

  6. Hematogones With Lambda Light Chain Restriction in a 4-Year-Old Boy With Burkitt Lymphoma: A Potential Diagnostic Pitfall

    PubMed Central

    Guillory, Tesha; Li, Shiyong; Bergsagel, Daniel J.; Weinzierl, Elizabeth; Bunting, Silvia T.

    2016-01-01

    Hematogones are immature normal B cell precursors with a characteristic immunophenotype profile on flow cytometry that typically do not express surface immunoglobulin light chains. In this report, we describe a case in which the hematogones exhibit light chain restriction. Our patient was a 4-year-old boy with a complicated medical history involving treatment for a presumed bilateral Wilms tumor of the kidney that on later resection was diagnosed as Burkitt lymphoma. Flow cytometry analysis of his bone marrow revealed a small distinct population of cells expressing dim cluster of differentiation (CD)10, CD19, CD22, CD38, dim CD58, human leukocyte antigen–D related (HLA-DR), and dim CD45, which are characteristic of hematogones. These cells, however, demonstrated dim surface immunoglobulin lambda light-chain restriction. Molecular study results for immunoglobulin heavy and kappa light-chain gene rearrangements were negative. We present this case to raise awareness of the potential pitfalls of working up bone marrow for involvement by B cell lymphoproliferative disorder. PMID:27069035

  7. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    SciTech Connect

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

  8. Light Chain Deposition Disease Presenting as Cholestatic Jaundice: A Case Report

    PubMed Central

    Kumar, Prasanna N.

    2012-01-01

    Light-chain deposition disease (LCDD) is characterized by tissue deposition of the immunoglobulin light chains in multiple organs. These deposits appear similar to amyloid on routine sections, but differ in their staining properties and ultrastructural appearance. The deposits of LCCD are non -Congophilic and do not exhibit a fibrillar ultrastructure; while, the proteinaceous substance seen in primary amyloidosis is Congo red positive and fibrillar. One of the most common organs to be involved in LCDD is the kidney. Earlier reports on cases of LCDD have mostly shown simultaneous liver and renal involvement, there are very few cases in the literature describing LCDD of the liver without renal involvement. This report describes a patient who presented with severe cholestatic jaundice and liver cell failure with normal renal function. PMID:22359728

  9. Method for altering antibody light chain interactions

    DOEpatents

    Stevens, Fred J.; Stevens, Priscilla Wilkins; Raffen, Rosemarie; Schiffer, Marianne

    2002-01-01

    A method for recombinant antibody subunit dimerization including modifying at least one codon of a nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in the interface segment of the light polypeptide variable region, the charged amino acid having a first polarity; and modifying at least one codon of the nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in an interface segment of the heavy polypeptide variable region corresponding to a position in the light polypeptide variable region, the charged amino acid having a second polarity opposite the first polarity. Nucleic acid sequences which code for novel light chain proteins, the latter of which are used in conjunction with the inventive method, are also provided.

  10. Expression of heavy chain-only antibodies can support B-cell development in light chain knockout chickens.

    PubMed

    Schusser, Benjamin; Collarini, Ellen J; Pedersen, Darlene; Yi, Henry; Ching, Kathryn; Izquierdo, Shelley; Thoma, Theresa; Lettmann, Sarah; Kaspers, Bernd; Etches, Robert J; van de Lavoir, Marie-Cecile; Harriman, William; Leighton, Philip A

    2016-09-01

    Since the discovery of antibody-producing B cells in chickens six decades ago, chickens have been a model for B-cell development in gut-associated lymphoid tissue species. Here we describe targeting of the immunoglobulin light chain locus by homologous recombination in chicken primordial germ cells (PGCs) and generation of VJCL knockout chickens. In contrast to immunoglobulin heavy chain knockout chickens, which completely lack mature B cells, homozygous light chain knockout (IgL(-/-) ) chickens have a small population of B lineage cells that develop in the bursa and migrate to the periphery. This population of B cells expresses the immunoglobulin heavy chain molecule on the cell surface. Soluble heavy-chain-only IgM and IgY proteins of reduced molecular weight were detectable in plasma in 4-week-old IgL(-/-) chickens, and antigen-specific IgM and IgY heavy chain proteins were produced in response to immunization. Circulating heavy-chain-only IgM showed a deletion of the CH1 domain of the constant region enabling the immunoglobulin heavy chain to be secreted in the absence of the light chain. Our data suggest that the heavy chain by itself is enough to support all the important steps in B-cell development in a gut-associated lymphoid tissue species.

  11. Loss of tubular creatinine secretion as the only sign of tubular proximal cell dysfunction in light chain proximal tubulopathy

    PubMed Central

    Stehlé, Thomas; Vignon, Marguerite; Flamant, Martin; Figueres, Marie-Lucile; Rabant, Marion; Rodenas, Anita; Noël, Laure-Hélène; Arnulf, Bertrand; Vidal-Petiot, Emmanuelle

    2016-01-01

    Abstract Light chain proximal tubulopathy (LCPT) is a rare disease, characterized by cytoplasmic inclusions of light chain (usually kappa) immunoglobulins. Clinical presentation is usually a Fanconi syndrome. The proximal tubular dysfunction can be incomplete, and exceptional cases of LCPT without any tubular dysfunction have even been described. Here, we report a case of LCPT in which the only sign of proximal tubulopathy is the absence of secretion of creatinine, as assessed by the simultaneous measurement of renal clearance of creatinine and 51CrEDTA. The loss of tubular creatinine secretion as a sign of tubular proximal cell dysfunction ought to be identified in patients with light chain proximal tubulopathy as it leads to a clinically relevant underestimation of GFR by the creatinine-derived equations. The prevalence and prognostic significance of this particular proximal tubular damage in LCPT remain to be determined. PMID:27367983

  12. Myosin, Transgelin, and Myosin Light Chain Kinase

    PubMed Central

    Léguillette, Renaud; Laviolette, Michel; Bergeron, Celine; Zitouni, Nedjma; Kogut, Paul; Solway, Julian; Kachmar, Linda; Hamid, Qutayba; Lauzon, Anne-Marie

    2009-01-01

    Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma. Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax. Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay. Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion. Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma. PMID:19011151

  13. [Clinical usefulness of serum free light chain measurement in monoclonal gammopathy].

    PubMed

    Shimazaki, Chihiro; Murakami, Hirokazu; Sawamura, Morio; Matsuda, Masayuki; Kinoshita, Tomohiro; Hata, Hiroyuki; Sugiura, Isamu; Tsushita, Keitaroh; Nagura, Eiichi; Kosugi, Hiroshi; Itoh, Junji; Shimizu, Kazuyuki

    2010-04-01

    Recently, a highly sensitive assay (FREELITE) capable of measuring serum immunoglobulin-free light chains (FLC) was developed. An abnormal kappa/lambda ratio supports the presence of clonal plasma cell expansion. Using this assay, we measured serum and urine samples of 178 healthy volunteers, 184 patients with polyclonal gamma-globulinemia and 150 patients with monoclonal gamma-globulinemia. The diagnostic sensitivity of the FLC assays for monoclonal gammopathies was 88.0% and the specificity for healthy volunteers and polyclonal gammopathies was 96.1%. The minimal detection sensitivity of this assay for serum FLC was 0.3 mg/l and was greater than 100-fold more sensitive than serum protein electrophoresis (SPE). The combination of FLC with SPE and immunoelectrophoresis identified 99% of patients with monoclonal gammopathies. Effective treatment often leads to a more rapid reduction of the involved FLC level relative to the intact immunoglobulin or total light chain concentration because the half-life of FLC is <6 hours. These observations suggest that FREELITE is useful for diagnosis, disease monitoring and assessment of response to treatment in patients with monoclonal gammopathies such as multiple myeloma and AL amyloidosis.

  14. Differential effects on light chain amyloid formation depend on mutations and type of glycosaminoglycans.

    PubMed

    Blancas-Mejía, Luis M; Hammernik, Jared; Marin-Argany, Marta; Ramirez-Alvarado, Marina

    2015-02-20

    Amyloid light chain (AL) amyloidosis is a protein misfolding disease where immunoglobulin light chains sample partially folded states that lead to misfolding and amyloid formation, resulting in organ dysfunction and death. In vivo, amyloid deposits are found in the extracellular space and involve a variety of accessory molecules, such as glycosaminoglycans, one of the main components of the extracellular matrix. Glycosaminoglycans are a group of negatively charged heteropolysaccharides composed of repeating disaccharide units. In this study, we investigated the effect of glycosaminoglycans on the kinetics of amyloid fibril formation of three AL cardiac amyloidosis light chains. These proteins have similar thermodynamic stability but exhibit different kinetics of fibril formation. We also studied single restorative and reciprocal mutants and wild type germ line control protein. We found that the type of glycosaminoglycan has a different effect on the kinetics of fibril formation, and this effect seems to be associated with the natural propensity of each AL protein to form fibrils. Heparan sulfate accelerated AL-12, AL-09, κI Y87H, and AL-103 H92D fibril formation; delayed fibril formation for AL-103; and did not promote any fibril formation for AL-12 R65S, AL-103 delP95aIns, or κI O18/O8. Chondroitin sulfate A, on the other hand, showed a strong fibril formation inhibition for all proteins. We propose that heparan sulfate facilitates the formation of transient amyloidogenic conformations of AL light chains, thereby promoting amyloid formation, whereas chondroitin sulfate A kinetically traps partially unfolded intermediates, and further fibril elongation into fibrils is inhibited, resulting in formation/accumulation of oligomeric/protofibrillar aggregates. PMID:25538238

  15. Recruitment of Light Chains by Homologous and Heterologous Fibrils Shows Distinctive Kinetic and Conformational Specificity.

    PubMed

    Blancas-Mejía, Luis M; Ramirez-Alvarado, Marina

    2016-05-31

    Light chain amyloidosis is a protein misfolding disease in which immunoglobulin light chains aggregate as insoluble fibrils that accumulate in extracellular deposits. Amyloid fibril formation in vitro has been described as a nucleation-polymerization, autocatalytic reaction in which nascent fibrils catalyze formation of new fibrils, recruiting soluble protein into the fibril. In this context, it is also established that preformed fibrils or "seeds" accelerate fibril formation. In some cases, seeds with a substantially different sequence are able to accelerate the reaction, albeit with a lower efficiency. In this work, we studied the recruitment and addition of monomers in the presence of seeds of five immunoglobulin light chain proteins, covering a broad range of protein stabilities and amyloidogenic properties. Our data reveal that in the presence of homologous or heterologous seeds, the fibril formation reactions become less stochastic than de novo reactions. The kinetics of the most amyloidogenic proteins (AL-T05 and AL-09) do not present significant changes in the presence of seeds. Amyloidogenic protein AL-103 presented fairly consistent acceleration with all seeds. In contrast, the less amyloidogenic proteins (AL-12 and κI) presented dramatic differential effects that are dependent on the kind of seed used. κI had a poor efficiency to elongate preformed fibrils. Together, these results indicate that fibril formation is kinetically determined by the conformation of the amyloidogenic precursor and modulated by the differential ability of each protein to either nucleate or elongate fibrils. We observe morphological and conformational properties of some seeds that do not favor elongation with some proteins, resulting in a delay in the reaction. PMID:27158939

  16. Unusual Presentation of Light Chain Deposition Disease: A Case Report

    PubMed Central

    Uppal, Mayank; Amitabh, Vindu; Agrawal, Usha

    2016-01-01

    Light Chain Deposition Disease (LCDD) is a rare disease characterized by deposition of monoclonal non-amyloid light chains in multiple organs. We report an unusual histologic manifestation of LCDD in a 55-year-old female patient, who presented with nephrotic syndrome and an increased serum creatinine. This case of LCDD had features of cast nephropathy on biopsy which is diagnostic of myeloma kidney, when the patient was clinically asymptomatic. Serum electrophoresis showed no abnormal band. There was no other evidence of a B-cell clonal disorder or amyloidosis. Following chemotherapy, improvement in renal function correlated with a reduction in circulating light-chain levels. PMID:27437235

  17. [The renal pathology in light chain deposition disease].

    PubMed

    Giannakakis, K N; Faraggiana, T

    2005-01-01

    Light Chain Deposition Disease (LCDD) is a relatively frequent renal disease associated with dysproteinemia. Although the light chain deposits can be widespread, the kidney is the most frequently involved organ, and renal involvement can dominate the clinical condition. The morphological features of LCDD can be recognized by light microscopy; however, the diagnosis can be made certain only by immunofluorescence microscopy, using antisera to kappa and lambda chains, and by electron microscopy.

  18. Myosin light chain genes in the turkey (Meleagris gallopavo).

    PubMed

    Chaves, L D; Ostroski, B J; Reed, K M

    2003-01-01

    Myosin light chains associate with the motor protein myosin and are believed to play a role in the regulation of its actin-based ATPase activity. Myosin light chain cDNA clones from the turkey (Meleagris gallopavo) were isolated and sequenced. One sequence corresponded to an alternative transcript, the skeletal muscle essential light chain (MYL1 isoform 1) and a second to the smooth muscle isoform of myosin light chain (MYL6). The DNA and predicted amino acid sequences of both light chain genes were compared to that of the chicken. Based on the cDNA sequence, oligonucleotide primers were designed to amplify genomic DNA from six of the seven introns of the MYL1 gene. Approximately 5 kb of DNA was sequenced (introns and 3' UTR) and evaluated for the presence of single nucleotide polymorphisms (SNPs). SNPs were verified by sequencing common intron regions from multiple individuals and three polymorphisms were used to genotype pedigreed families. MYL1 is assigned to a turkey linkage group that corresponds to a region of chicken chromosome 7 (GGA7). The results of this study provide genomic reagents for comparative studies of avian muscle components and muscle biology.

  19. Immunoglobulin synthesis in primary and myeloma amyloidosis.

    PubMed Central

    Preud'homme, J L; Ganeval, D; Grünfeld, J P; Striker, L; Brouet, J C

    1988-01-01

    Bone marrow cells from 14 patients with primary amyloidosis and two patients with myeloma amyloidosis were studied by immunofluorescence and biosynthesis experiments after incorporation of radioactive amino acids. Cells from four patients affected with non-myeloma secondary amyloidosis were also studied as controls. In primary amyloidosis, monoclonal plasma cell populations were demonstrated by immunofluorescence in virtually every case, even in patients without serum and urine monoclonal immunoglobulin and with a normal percentage of bone marrow plasma cells. Biosynthesis experiments showed the secretion of large amounts of free light chains, most often of the lambda type, in every primary or myeloma amyloidosis case and the presence of light chain fragments in almost all cases. Special features in certain patients were the synthesis of short gamma chains (two cases), assembly block at the HL half molecule level of a monoclonal IgA (one case) and secretion of decameric abnormally large kappa chains (one case). This is in contrast with non-myelomatous secondary amyloidosis where the distribution of bone marrow plasma cells was normal by immunofluorescence and where normal sized immunoglobulins were synthesized, without free light chain secretion and fragments. These data confirm that primary amyloidosis belongs to plasma cell dyscrasias and emphasize the role of free light chains and light chain fragments in the pathogenesis of amyloid deposition. PMID:3145161

  20. Multiple myeloma-associated skin light chain amyloidosis: A case of misdiagnosis

    PubMed Central

    XIAO, LI; LIN, FENGXIA; XIAO, RONG; HU, CHUN; DENG, MINGYANG; LI, DAIQIANG; SHE, XIAOLING; LIU, FUYOU; SUN, LIN

    2016-01-01

    The present study reports the case of a 42-year-old male with multiple myeloma (MM)-associated skin light chain amyloidosis who presented with skin purpura as the initial symptom, which was misdiagnosis as Henoch-Schönlein purpura nephritis prior to admission to the Second Xiangya Hospital (Changsha, Hunan, China). The patient presented with purpura, papules petechiae and spontaneous ecchymosis, which was located scattered around the neck, chest and limbs, accompanied by a small amount of bleeding in the conjunctival and oral mucosa, and a swollen tongue. Upon laboratory examination, the serum immunological change showed increased serum immunoglobulin G and λ light chain levels, and a urine Bence Jones protein level of >1 g/24 h. This was accompanied with an abnormal result for immunofixation electrophoresis, and positive staining with Congo red showing apple-green birefringence in skin biopsy specimens. Thus, the patient was diagnosed with MM-associated skin amyloidosis with the initial symptom of skin purpura. Following treatment with chemotherapy consisting of prednisone and bortezomib, the skin lesions markedly improved. The present study indicates that the presentation of skin purpura in systemic amyloidosis associated with MM may be an important aid in the diagnosis and direct treatment of this disease in the clinic. PMID:27284363

  1. Combined use of free light chain and heavy/light chain ratios allow diagnosis and monitoring of patients with monoclonal gammopathies: Experience of a single institute, with three exemplar case reports

    PubMed Central

    Gagliardi, Alfredo; Carbone, Claudio; Russo, Angela; Cuccurullo, Rosanna; Lucania, Anna; Cioppa, Paola Della; Misso, Gabriella; Caraglia, Michele; Tommasino, Catello; Mastrullo, Lucia

    2016-01-01

    Monoclonal gammopathies are characterized by serum monoclonal component (MC) plus an intact immunoglobulin and a free light chain (FLC), or a combination of both. The measurement of FLC with Freelite® is the standard practice recommended by International Myeloma Working Group guidelines. Recently, Hevylite® heavy/light chains (HLC) assays were introduced to specifically target junctional epitopes between the heavy and light chains of intact immunoglobulins, allowing the independent quantification of the involved (MC) and uninvolved (polyclonal immunoglobulin background) HLC isotype. Between January 2012 and March 2014, 90 patients were examined: 49 multiple myeloma (MM), 6 smoldering MM (SMM) and 35 monoclonal gammopathy of undetermined significance (MGUS). Of these 90 patients, 300 samples were collected at different times. The diagnostic and monitoring contribution of Hevylite A and G assays was assessed in all 90 patients examined. Additionally, 3 representative cases were selected. The Hevylite absolute values and ratio demonstrated high sensitivity and specificity with respect to serum protein electrophoresis and serum immunofixation. The combined use of Hevylite A and G with Freelite was particularly useful in dubious cases with more than one MC or with co-migrating components, as well as in the course of monitoring to assess the independent change of FLC and HLC, possibly reflecting the presence of clonal heterogeneity in the cohort. From this study, it can be concluded that FLC and HLC are independent, useful markers to monitor the MC and to assess with greater specificity and sensitivity the effect of therapy, thereby providing clinical support. Further studies are required to assess the prognostic potential of Hevylite in MGUS and SMM.

  2. Combined use of free light chain and heavy/light chain ratios allow diagnosis and monitoring of patients with monoclonal gammopathies: Experience of a single institute, with three exemplar case reports

    PubMed Central

    Gagliardi, Alfredo; Carbone, Claudio; Russo, Angela; Cuccurullo, Rosanna; Lucania, Anna; Cioppa, Paola Della; Misso, Gabriella; Caraglia, Michele; Tommasino, Catello; Mastrullo, Lucia

    2016-01-01

    Monoclonal gammopathies are characterized by serum monoclonal component (MC) plus an intact immunoglobulin and a free light chain (FLC), or a combination of both. The measurement of FLC with Freelite® is the standard practice recommended by International Myeloma Working Group guidelines. Recently, Hevylite® heavy/light chains (HLC) assays were introduced to specifically target junctional epitopes between the heavy and light chains of intact immunoglobulins, allowing the independent quantification of the involved (MC) and uninvolved (polyclonal immunoglobulin background) HLC isotype. Between January 2012 and March 2014, 90 patients were examined: 49 multiple myeloma (MM), 6 smoldering MM (SMM) and 35 monoclonal gammopathy of undetermined significance (MGUS). Of these 90 patients, 300 samples were collected at different times. The diagnostic and monitoring contribution of Hevylite A and G assays was assessed in all 90 patients examined. Additionally, 3 representative cases were selected. The Hevylite absolute values and ratio demonstrated high sensitivity and specificity with respect to serum protein electrophoresis and serum immunofixation. The combined use of Hevylite A and G with Freelite was particularly useful in dubious cases with more than one MC or with co-migrating components, as well as in the course of monitoring to assess the independent change of FLC and HLC, possibly reflecting the presence of clonal heterogeneity in the cohort. From this study, it can be concluded that FLC and HLC are independent, useful markers to monitor the MC and to assess with greater specificity and sensitivity the effect of therapy, thereby providing clinical support. Further studies are required to assess the prognostic potential of Hevylite in MGUS and SMM. PMID:27698801

  3. Antibody elbow angles are influenced by their light chain class

    SciTech Connect

    Stanfield, R; Zemla, A; Wilson, I; Rupp, B

    2006-01-12

    We have examined the elbow angles for 365 different Fab fragments, and observe that Fabs with lambda light chains have adopted a wider range of elbow angles than their kappa-chain counterparts, and that the lambda light chain Fabs are frequently found with very large (>195{sup o}) elbow angles. This apparent hyperflexibility of lambda-chain Fabs may be due to an insertion in their switch region, which is one residue longer than in kappa chains, with glycine occurring most frequently at the insertion position. A new, web-based computer program that was used to calculate the Fab elbow angles is also described.

  4. Genomic clone for sandbar shark lambda light chain: generation of diversity in the absence of gene rearrangement.

    PubMed Central

    Hohman, V S; Schuchman, D B; Schluter, S F; Marchalonis, J J

    1993-01-01

    While the general structure of immunoglobulin chains has remained relatively unchanged throughout evolution, the organization of the genes encoding these molecules differs substantially. To understand how the rearranging immunoglobulin system arose, it is necessary to examine living representatives of the most early vertebrate phyla. Elasmo-branches, which include the sharks, skates, and rays, are the most primitive phylogenetic class of vertebrates from which immunoglobulin DNA sequences have been obtained. In the sandbar shark (Carcharhinus plumbeus), the genes are arranged in individual clusters in which a single variable (V), joining (J), and constant (C) region gene, along with upstream regulatory elements, span a distance of approximately 4.4 kb or approximately 5.8 kb. We report the complete sequence of a genomic clone encoding sandbar shark lambda light chain. A unique finding of our study is that the V and J genes are fused in the germ line. Three additional clones have been shown by DNA sequencing to also have fused V and J genes. The four clones have complementarity-determining regions 3 of various lengths and amino acid sequence variability similar to the products of rearranged genes. Furthermore, analysis by polymerase chain reaction technology revealed an additional 26 genomic clones demonstrating fusion of the V and J segments. Therefore, VJ fusion is the prominent organizational feature of sandbar shark immunoglobulin light chain genes. This finding raises questions concerning the necessity of recombination to produce an antibody repertoire capable of reacting against a diverse array of antigens. Images Fig. 1 Fig. 2 Fig. 3 PMID:8234330

  5. Tertiary structure of human {Lambda}6 light chains.

    SciTech Connect

    Pokkuluri, P. R.; Solomon, A.; Weiss, D. T.; Stevens, F. J.; Schiffer, M.; Center for Mechanistic Biology and Biotechnology; Univ. of Tennessee Medical Center /Graduate School of Medicine

    1999-01-01

    AL amyloidosis is a disease process characterized by the pathologic deposition of monoclonal light chains in tissue. To date, only limited information has been obtained on the molecular features that render such light chains amyloidogenic. Although protein products of the major human V kappa and V lambda gene families have been identified in AL deposits, one particular subgroup--lambda 6--has been found to be preferentially associated with this disease. Notably, the variable region of lambda 6 proteins (V lambda 6) has distinctive primary structural features including the presence in the third framework region (FR3) of two additional amino acid residues that distinguish members of this subgroup from other types of light chains. However, the structural consequences of these alterations have not been elucidated. To determine if lambda 6 proteins possess unique tertiary structural features, as compared to light chains of other V lambda subgroups, we have obtained x-ray diffraction data on crystals prepared from two recombinant V lambda 6 molecules. These components, isolated from a bacterial expression system, were generated from lambda 6-related cDNAs cloned from bone marrow-derived plasma cells from a patient (Wil) who had documented AL amyloidosis and another (Jto) with multiple myeloma and tubular cast nephropathy, but no evident fibrillar deposits. The x-ray crystallographic analyses revealed that the two-residue insertion located between positions 68 and 69 (not between 66 and 67 as previously surmised) extended an existing loop region that effectively increased the surface area adjacent to the first complementarity determining region (CDR1). Further, an unusual interaction between the Arg 25 and Phe 2 residues commonly found in lambda 6 molecules was noted. However, the structures of V lambda 6 Wil and Jto also differed from each other, as evidenced by the presence in the latter of certain ionic and hydrophobic interactions that we posit increased protein

  6. Cysteine Racemization on IgG Heavy and Light Chains

    PubMed Central

    Zhang, Qingchun; Flynn, Gregory C.

    2013-01-01

    Under basic pH conditions, the heavy chain 220-light chain 214 (H220-L214) disulfide bond, found in the flexible hinge region of an IgG1, can convert to a thioether. Similar conditions also result in racemization of the H220 cysteine. Here, we report that racemization occurs on both H220 and L214 on an IgG1 with a λ light chain (IgG1λ) but almost entirely on H220 of an IgGl with a κ light chain (IgG1κ) under similar conditions. Likewise, racemization was detected at significant levels on H220 and L214 on endogenous human IgG1λ but only at the H220 position on IgG1κ. Low but measurable levels of d-cysteines were found on IgG2 cysteines in the hinge region, both with monoclonal antibodies incubated under basic pH conditions and on antibodies isolated from human serum. A simplified reaction mechanism involving reversible β-elimination on the cysteine is presented that accounts for both base-catalyzed racemization and thioether formation at the hinge disulfide. PMID:24142697

  7. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    SciTech Connect

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; Ramirez-Alvarado, Marina; Donohoe, Dallas; Williams, Angela; Macy, Sallie; Wooliver, Craig; Wortham, Dale; Morrell-Falvey, Jennifer; Foster, Carmen M.; Kennel, Stephen J.; Wall, Jonathan S.

    2015-09-22

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. Ultimately, these data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

  8. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    DOE PAGES

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; Ramirez-Alvarado, Marina; Donohoe, Dallas; Williams, Angela; Macy, Sallie; Wooliver, Craig; Wortham, Dale; Morrell-Falvey, Jennifer; et al

    2015-09-22

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presencemore » of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. Ultimately, these data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.« less

  9. Role of myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability in vitro and in vivo.

    PubMed

    Wu, Fan; Guo, Xiaohua; Xu, Jing; Wang, Weiju; Li, Bingling; Huang, Qiaobing; Su, Lei; Xu, Qiulin

    2016-03-01

    We have previously reported that advanced glycation end products activated Rho-associated protein kinase and p38 mitogen-activated protein kinase, causing endothelial hyperpermeability. However, the mechanisms involved were not fully clarified. Here, we explored the role of myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability. Myosin light chain phosphorylation significantly increased by advanced glycation end products in endothelial cells in a time- and dose-dependent manner, indicating that myosin light chain phosphorylation is involved in the advanced glycation end product pathway. Advanced glycation end products also induced myosin phosphatase-targeting subunit 1 phosphorylation, and small interfering RNA knockdown of the receptor for advanced glycation end products, or blocking myosin light chain kinase with its inhibitor, ML-7, or small interfering RNA abated advanced glycation end product-induced myosin light chain phosphorylation. Advanced glycation end product-induced F-actin rearrangement and endothelial hyperpermeability were also diminished by inhibition of receptor for advanced glycation end product or myosin light chain kinase signalling. Moreover, inhibiting myosin light chain kinase with ML-7 or blocking receptor for advanced glycation end product with its neutralizing antibody attenuated advanced glycation end product-induced microvascular hyperpermeability. Our findings suggest a novel role for myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability.

  10. Constitutive phosphorylation of cardiac myosin regulatory light chain in vivo.

    PubMed

    Chang, Audrey N; Battiprolu, Pavan K; Cowley, Patrick M; Chen, Guohua; Gerard, Robert D; Pinto, Jose R; Hill, Joseph A; Baker, Anthony J; Kamm, Kristine E; Stull, James T

    2015-04-24

    In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca(2+) sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity.

  11. Serologically defined V region subgroups of human lambda light chains.

    PubMed

    Solomon, A; Weiss, D T

    1987-08-01

    The availability of numerous antisera prepared against lambda-type Bence Jones proteins and lambda chains of known amino acid sequence has led to the differentiation and classification of human lambda light chains into one of five V lambda subgroups. The five serologically defined subgroups, V lambda I, V lambda II, V lambda III, V lambda IV, and V lambda VI, correspond to the chemical classification that is based on sequence homologies in the first framework region (FR1). Proteins designated by sequence as lambda V react with specific anti-lambda II antisera and are thus included in the V lambda II subgroup classification. The isotypic nature of the five V lambda subgroups was evidenced through analyses of lambda-type light chains that were isolated from the IgG of normal individuals. Based on analyses of 116 Bence Jones proteins, the frequency of distribution of the lambda I, lambda II/V, lambda III, lambda IV, and lambda VI proteins in the normal lambda chain population is estimated to be 27%, 37%, 23%, 3%, and 10%, respectively. This distribution of V lambda subgroups was comparable to that found among 82 monoclonal Ig lambda proteins. Considerable V lambda intragroup antigenic heterogeneity was also apparent. At least two sub-subgroups were identified among each of the five major V lambda subgroups, implying the existence of multiple genes in the human V lambda genome. The V lambda classification of 54 Ig lambda proteins obtained from patients with primary or multiple myeloma-associated amyloidosis substantiated the preferential association of lambda VI light chains with amyloidosis AL and the predominance of the normally rare V lambda VI subgroup in this disease. PMID:3110284

  12. Phenotypic, transcriptomic, and genomic features of clonal plasma cells in light-chain amyloidosis.

    PubMed

    Paiva, Bruno; Martinez-Lopez, Joaquin; Corchete, Luis A; Sanchez-Vega, Beatriz; Rapado, Inmaculada; Puig, Noemi; Barrio, Santiago; Sanchez, Maria-Luz; Alignani, Diego; Lasa, Marta; García de Coca, Alfonso; Pardal, Emilia; Oriol, Alberto; Garcia, Maria-Esther Gonzalez; Escalante, Fernando; González-López, Tomás J; Palomera, Luis; Alonso, José; Prosper, Felipe; Orfao, Alberto; Vidriales, Maria-Belen; Mateos, María-Victoria; Lahuerta, Juan-Jose; Gutierrez, Norma C; San Miguel, Jesús F

    2016-06-16

    Immunoglobulin light-chain amyloidosis (AL) and multiple myeloma (MM) are 2 distinct monoclonal gammopathies that involve the same cellular compartment: clonal plasma cells (PCs). Despite the fact that knowledge about MM PC biology has significantly increased in the last decade, the same does not apply for AL. Here, we used an integrative phenotypic, molecular, and genomic approach to study clonal PCs from 24 newly diagnosed patients with AL. Through principal-component-analysis, we demonstrated highly overlapping phenotypic profiles between AL and both monoclonal gammopathy of undetermined significance and MM PCs. However, in contrast to MM, highly purified fluorescence-activated cell-sorted clonal PCs from AL (n = 9) showed almost normal transcriptome, with only 38 deregulated genes vs normal PCs; these included a few tumor-suppressor (CDH1, RCAN) and proapoptotic (GLIPR1, FAS) genes. Notwithstanding, clonal PCs in AL (n = 11) were genomically unstable, with a median of 9 copy number alterations (CNAs) per case, many of such CNAs being similar to those found in MM. Whole-exome sequencing (WES) performed in 5 AL patients revealed a median of 15 nonrecurrent mutations per case. Altogether, our results show that in the absence of a unifying mutation by WES, clonal PCs in AL display phenotypic and CNA profiles similar to MM, but their transcriptome is remarkably similar to that of normal PCs. PMID:27069257

  13. Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains.

    SciTech Connect

    Dul, J. L.; Davis, D. P.; Williamson, E. K.; Stevens, F. J.; Argon, Y.; Univ. of Chicago

    2001-02-19

    In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its {beta} sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocated out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach.

  14. Myosin light chain kinase (MLCK) regulates cell migration in a myosin regulatory light chain phosphorylation-independent mechanism.

    PubMed

    Chen, Chen; Tao, Tao; Wen, Cheng; He, Wei-Qi; Qiao, Yan-Ning; Gao, Yun-Qian; Chen, Xin; Wang, Pei; Chen, Cai-Ping; Zhao, Wei; Chen, Hua-Qun; Ye, An-Pei; Peng, Ya-Jing; Zhu, Min-Sheng

    2014-10-10

    Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.

  15. Successful Treatment of Amyloid Light-chain Amyloidosis in a Charcot-Marie-Tooth Disease Patient with Lenalidomide, Cyclophosphamide, and Dexamethasone.

    PubMed

    Kikukawa, Yoshitaka; Hata, Hiroyuki; Ueda, Mitsuharu; Yamashita, Taro; Nasu, Singo; Ide, Kazuhiko; Ueno, Shikiko; Ando, Yukio; Mitsuya, Hiroaki; Okuno, Yutaka

    2016-01-01

    A 70-year-old woman with Charcot-Marie-Tooth disease (CMT) suffered from nephrotic syndrome and a renal biopsy revealed non-AA amyloid depositions that contained immunoglobulin light chain λ. Her serum λ free LC was elevated to 80.8 mg/L and she was diagnosed with primary amyloid light-chain (AL) amyloidosis. She was subsequently treated with lenalidomide, cyclophosphamide, and dexamethasone (RCD). After 14 cycles of RCD, she achieved complete remission. Her serum albumin levels gradually normalized to 3.1 g/dL. No exacerbation of neurologic symptoms related to CMT was observed. Thus, RCD may be a well-tolerated and effective regimen for treating AL amyloidosis in patients with CMT disease. PMID:27629972

  16. In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study

    PubMed Central

    Ami, Diletta; Lavatelli, Francesca; Rognoni, Paola; Palladini, Giovanni; Raimondi, Sara; Giorgetti, Sofia; Monti, Luca; Doglia, Silvia Maria; Natalello, Antonino; Merlini, Giampaolo

    2016-01-01

    Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level. To this aim, we used Fourier transform infrared (FTIR) microspectroscopy for studying in situ unfixed tissues (heart and subcutaneous abdominal fat) from patients affected by AL amyloidosis. We compared the infrared response of affected tissues with that of ex vivo and in vitro fibrils obtained from the pathogenic LC derived from one patient, as well as with that of non amyloid-affected tissues. We demonstrated that the IR marker band of intermolecular β-sheets, typical of protein aggregates, can be detected in situ in LC amyloid-affected tissues, and that FTIR microspectroscopy allows exploring the inter- and intra-sample heterogeneity. We extended the infrared analysis to the characterization of other biomolecules embedded within the amyloid deposits, finding an IR pattern that discloses a possible role of lipids, collagen and glycosaminoglycans in amyloid deposition in vivo. PMID:27373200

  17. Quantification of β region IgA paraproteins - should we include immunochemical "heavy/light chain" measurements? Counterpoint.

    PubMed

    Paolini, Lucia

    2016-06-01

    Serum protein electrophoresis (SPE), serum immunofixation (s-IFE), free light chain measurement (FLC) and nephelometric measurements of total immunoglobulin in serum (IgTot) are some of the laboratory tests required for the management of plasma cell proliferative disorders. The monoclonal protein is usually visible on SPE as a spike (M-spike) in the γ region and the derived densitogram is used to quantify it relative to serum total protein concentration. IgA M-protein, however, often migrates in the β region on SPE and its quantification can be masked by other serum proteins that migrate in this region. The immunoassay Hevylite™ (heavy/light chain, HLC) seems to solve this problem: it quantifies the involved/uninvolved isotype, calculating the ratio IgAκ/IgAλ, considered indicative of clonal proliferation. However, this test seems redundant in the case of artifacts on SPE such as obvious hemolysis or lipemia, or if the IgA M-spike is clearly visible in the β region. In conclusion whereas the IgA HLC assay does not represent an alternative to SPE and s-IFE in the diagnostic patient workup, it may prove to be an alternative to SPE, s-IFE and total IgA quantification in risk stratification and evaluation of response to therapy in patients affected by MM and other monoclonal plasma proliferative disorders.

  18. In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study.

    PubMed

    Ami, Diletta; Lavatelli, Francesca; Rognoni, Paola; Palladini, Giovanni; Raimondi, Sara; Giorgetti, Sofia; Monti, Luca; Doglia, Silvia Maria; Natalello, Antonino; Merlini, Giampaolo

    2016-01-01

    Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level. To this aim, we used Fourier transform infrared (FTIR) microspectroscopy for studying in situ unfixed tissues (heart and subcutaneous abdominal fat) from patients affected by AL amyloidosis. We compared the infrared response of affected tissues with that of ex vivo and in vitro fibrils obtained from the pathogenic LC derived from one patient, as well as with that of non amyloid-affected tissues. We demonstrated that the IR marker band of intermolecular β-sheets, typical of protein aggregates, can be detected in situ in LC amyloid-affected tissues, and that FTIR microspectroscopy allows exploring the inter- and intra-sample heterogeneity. We extended the infrared analysis to the characterization of other biomolecules embedded within the amyloid deposits, finding an IR pattern that discloses a possible role of lipids, collagen and glycosaminoglycans in amyloid deposition in vivo. PMID:27373200

  19. Variable domain structure of {kappa}IV human light chain len : high homology to the murine light chain McPC603.

    SciTech Connect

    Huang, D.-B.; Chang, C.-H.; Ainsworth, C.; Johnson, G.; Solomon, A.; Stevens, F. J.; Schiffer, M.; Center for Mechanistic Biology and Biotechnology; Univ. of Tennessee Medical Center

    1997-12-01

    Antibody light chains of the {kappa} subgroup are the predominant light chain component in human immune responses and are used almost exclusively in the antibody repertoire of mice. Human {kappa} light chains comprise four subgroups. To date, all crystallographic studies of human {kappa} light chains were carried out on proteins of the {kappa}I subgroup. The light chain produced by multiple myeloma patient Len, was of the {kappa}IV subgroup, it differed by only one residue from the germ-line gene encoded protein. The variable domain fragment of the light chain was crystallized from ammonium sulfate in space group C222{sub 1}. The crystal structure was determined by molecular replacement and refined at 1.95 Angstrom resolution to an R-factor of 0.15. Protein Len has six additional residues in its CDR1 segment compared to the {kappa}I proteins previously characterized. The {kappa}IV variable domain. Len, differs in only 23 of 113 residues from murine {kappa} light chain McPC603. The RMS deviation upon superimposing their {alpha}-carbons was 0.69 Angstrom. The CDR1 segment of the human and murine variable domains have the same length and conformation although their amino acid sequences differ in 5 out of 17 residues. Structural features were identified that could account for the significantly higher stability of the human {kappa}IV protein relative to its murine counterpart. This human {kappa}IV light chain structure is the closest human homolog to a murine light chain and can be expected to facilitate detailed structural comparisons necessary for effective humanization of murine antibodies.

  20. B Meson Decays to mega K*, omega rho, omega omega, omega phi, and omega f0

    SciTech Connect

    Aubert, B.; Barate, R.; Bona, M.; Boutigny, D.; Couderc, F.; Karyotakis, Y.; Lees, J.P.; Poireau, V.; Tisserand, V.; Zghiche, A.; Grauges, E.; Palano, A.; Chen, J.C.; Qi, N.D.; Rong, G.; Wang, P.; Zhu, Y.S.; Eigen, G.; Ofte, I.; Stugu, B.; Abrams, G.S.; /LBL, Berkeley /UC, Berkeley /Birmingham U. /Ruhr U., Bochum /Bristol U. /British Columbia U. /Brunel U. /Novosibirsk, IYF /UC, Irvine /UCLA /UC, Riverside /UC, San Diego /UC, Santa Barbara /UC, Santa Cruz /Caltech /Cincinnati U. /Colorado U. /Colorado State U. /Dortmund U. /Dresden, Tech. U. /Ecole Polytechnique /Edinburgh U. /Ferrara U. /INFN, Ferrara /Frascati /Genoa U. /INFN, Genoa /Harvard U. /Heidelberg U. /Imperial Coll., London /Iowa U. /Iowa State U. /Johns Hopkins U. /Karlsruhe U., EKP /LLNL, Livermore /Liverpool U. /Queen Mary, U. of London /Royal Holloway, U. of London /Louisville U. /Manchester U. /Maryland U. /Massachusetts U., Amherst /MIT, LNS /McGill U. /Milan U. /INFN, Milan /Mississippi U. /Montreal U. /Mt. Holyoke Coll. /Naples U. /INFN, Naples /NIKHEF, Amsterdam /Notre Dame U. /Ohio State U. /Oregon U. /Padua U. /INFN, Padua /Paris U., VI-VII /Pennsylvania U. /Perugia U. /INFN, Perugia /Pisa U. /INFN, Pisa /Prairie View A-M /Princeton U. /Rome U. /INFN, Rome /Rostock U. /Rutherford /DAPNIA, Saclay /South Carolina U. /SLAC /Stanford U., Phys. Dept. /SUNY, Stony Brook /Tennessee U. /Texas U. /Texas U., Dallas /Turin U. /INFN, Turin /Trieste U. /INFN, Trieste /Valencia U., IFIC /Victoria U. /Warwick U. /Wisconsin U., Madison /Yale U. /Clermont-Ferrand U. /Basilicata U., Potenza

    2006-07-28

    The authors describe searches for B meson decays to the charmless vector-vector final states {omega}K*, {omega}p, {omega}{omega}, and {omega}{phi} with 233 x 10{sup 6} B{bar B} pairs produced in e{sup +}e{sup -} annihilation at {radical}s = 10.58 GeV. They also search for the vector-scalar B decay to {omega}f{sub 0}.

  1. Polymerization of immunoglobulin domains: A model system for the development of facilitated macromolecular assembly

    SciTech Connect

    Stevens, F.J.; Myatt, E.A.

    1991-01-01

    We have recently determined that monoclonal immunoglobulin light chains (Bence Jones proteins) are capable of reversible polymerization at room temperature. This property, as exhibited by immunoglobulin light chains (normally a component of an intact antibody molecule), may have novel implications for the development of molecular nanotechnology.'' The polymerization capability of the immunoglobulin light chain is associated with the so-called variable domain of this molecule. The variable domain is a durable, compact beta-sheet structure of molecular weight approximately 12,000. Most of the primary sequence variation is limited to one portion of the molecule, that portion associated with the contribution of immunoglobulin light chains to the recognition and binding of thousand of different antigens by antibodies. As a consequence of these variations, different light chains polymerize with different degrees of avidity, from negligible to extensive. The polymerization process depends on solution parameters such as Ph. Thus, polymerization might be induced at one pH and suppressed or reversed at another. Combinations of molecules of appropriate specificities could assemble into structures of predetermined three-dimensional forms and properties. These features suggest that Bence Jones proteins represent a powerful model system within which to develop empirical rules relevant to a technology of protein-based construction''. Development of these rules will require the combined efforts of biophysical and crystallographic studies, protein engineering, and molecular modeling. 53 refs., 5 figs.

  2. Polymerization of immunoglobulin domains: A model system for the development of facilitated macromolecular assembly

    SciTech Connect

    Stevens, F.J.; Myatt, E.A.

    1991-12-31

    We have recently determined that monoclonal immunoglobulin light chains (Bence Jones proteins) are capable of reversible polymerization at room temperature. This property, as exhibited by immunoglobulin light chains (normally a component of an intact antibody molecule), may have novel implications for the development of ``molecular nanotechnology.`` The polymerization capability of the immunoglobulin light chain is associated with the so-called variable domain of this molecule. The variable domain is a durable, compact beta-sheet structure of molecular weight approximately 12,000. Most of the primary sequence variation is limited to one portion of the molecule, that portion associated with the contribution of immunoglobulin light chains to the recognition and binding of thousand of different antigens by antibodies. As a consequence of these variations, different light chains polymerize with different degrees of avidity, from negligible to extensive. The polymerization process depends on solution parameters such as Ph. Thus, polymerization might be induced at one pH and suppressed or reversed at another. Combinations of molecules of appropriate specificities could assemble into structures of predetermined three-dimensional forms and properties. These features suggest that Bence Jones proteins represent a powerful model system within which to develop empirical rules relevant to a technology of protein-based ``construction``. Development of these rules will require the combined efforts of biophysical and crystallographic studies, protein engineering, and molecular modeling. 53 refs., 5 figs.

  3. The ferritin light-chain homologue promoter in Aedes aegypti.

    PubMed

    Pham, D Q-D; Chavez, C A

    2005-06-01

    Promoters that direct the expression of antipathogenic molecules to primary sites of pathogenic invasions provide a means to interfere with these invasions. Thus, they have the potential to be used in mosquito control. However, exogenous elements are known to lower the fitness of most insects, and given the ability of insects to evolve rapidly, all currently known promoters could be rendered useless. As transgenic mosquitoes may be a major component in the fight against mosquito-borne diseases, the identification of new mosquito promoters is needed. The promoter of the Aedes aegypti ferritin light-chain homologue (LCH) gene, a gene whose expression is induced in gut tissues during blood feeding has been identified and mapped. Transfection data indicate that the ferritin LCH promoter is a strong promoter. DNase I footprinting data and Transfac analyses suggest that the ferritin LCH promoter contains putative GATA, E2F, NIT2, TATA and DPE sites. These data together provide the first detailed map of a known ferritin LCH gene.

  4. Cargo selection by specific kinesin light chain 1 isoforms

    PubMed Central

    Woźniak, Marcin J; Allan, Victoria J

    2006-01-01

    Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures. PMID:17093494

  5. Immunoglobulin gene diversification in cattle.

    PubMed

    Meyer, A; Parng, C L; Hansal, S A; Osborne, B A; Goldsby, R A

    1997-01-01

    Research in several species has revealed that different types of mammals have evolved divergent molecular and cellular strategies for generating immunoglobulin diversity. Other chapters in this text have highlighted the specific characteristics unique to chicken, rabbit, mouse, human and sheep B lymphocyte development; namely indicating differences in the mechanisms of diversity and the site of primary B cell development. Studies of the bovine system have indicated that like the sheep system, the ileal Peyer's patch (IPP) is a likely chicken bursal equivalent, and is a site of primary B lymphocyte development. Substantial investigation in sheep has indicated that Ig diversity is created by untemplated somatic mutation and intense selective pressure (Reynaud et al., 1991). The frequency of alteration in the sheep Ig light chain gene locus also is characteristic of the bovine system, however, recent evidence from sequencing of bovine lambda light chain genes indicates that one mechanism that contributes to diversity is gene conversion, utilizing several pseudogenes located in the Ig locus (Parng et al., 1996). The mechanism by which this hyperalteration of Ig genes occurs in both sheep and cattle is poorly understood and is thus the focus of considerable investigation. The study of events in the IPP may also have informative ramifications for secondary diversification of the Ig repertoire by somatic hyperalteration in germinal centers.

  6. Recurrent Light Chain Proximal Tubulopathy in a Kidney Allograft.

    PubMed

    Angioi, Andrea; Amer, Hatem; Fervenza, Fernando C; Sethi, Sanjeev

    2016-09-01

    We describe a rare case of light chain proximal tubulopathy developing in a kidney transplant 12 months following transplantation. The patient was known to have a monoclonal gammopathy of undetermined significance (MGUS) for more than 15 years. A kidney biopsy done to determine the cause of decline in kidney transplant function showed light chain proximal tubulopathy characterized by numerous eosinophilic and fuchsinophilic granules in proximal tubular epithelial cells, which stained for κ light chains on pronase-based immunofluorescence studies. Electron microscopy confirmed the diagnosis and showed numerous amorphous and geometrically shaped inclusions in proximal tubular epithelial cells. Evaluation of free light chains revealed markedly elevated κ light chains and bone marrow biopsy showed 5% to 10% κ light chain-restricted plasma cells. Retrospective evaluation of the native kidney biopsy performed 15 years earlier also showed numerous fuchsinophilic granules in proximal tubules that stained brightly for κ light chains on pronase-based immunofluorescence studies. The patient was treated with a regimen of bortezomib and dexamethasone with good partial hematologic response and improvement of kidney function. To summarize, we describe a case of recurrent light chain proximal tubulopathy in the transplant, which is an unusual but important cause of decreased kidney function in the setting of a monoclonal gammopathy. PMID:27321964

  7. Structure and diversity of Mexican axolotl lambda light chains.

    PubMed

    André, S; Guillet, F; Charlemagne, J; Fellah, J S

    2000-11-01

    We report here the structure of cDNA clones encoding axolotl light chains of the lambda type. A single IGLC gene and eight different potential IGLV genes belonging to four different families were detected. The axolotl Cgamma domain has several residues or stretches of residues that are typically conserved in mammalian, avian, and Xenopus Cgamma, but the KATLVCL stretch, which is well conserved in the Cgamma and T-cell receptor Cbeta domains of many vertebrate species, is not well conserved. All axolotl Vgamma sequences closely match several human and Xenopus Vgamma-like sequences and, although the axolotl Cgamma and Vgamma sequences are very like their tetrapod homologues, they are not closely related to nontetrapod L chains. Southern blot experiments suggested the presence of a single IGLC gene and of a limited number of IGLV genes, and analysis of IGLV-J junctions clearly indicated that at least three of the IGLJ segments can associate with IGLV1, IGLV2, or IGLV3 subgroup genes. The overall diversity of the axolotl Vgamma CDR3 junctions seems to be of the same order as that of mammalian Vgamma chains. However, a single IGLV4 segment was found among the 45 cDNAs analyzed. This suggests that the axolotl IGL locus may have a canonical tandem structure, like the mammalian IGK or IGH loci. Immunofluorescence, immunoblotting, and microsequencing experiments strongly suggested that most, if not all L chains are of the gamma type. This may explain in part the poor humoral response of the axolotl. PMID:11132150

  8. Involvement of myosin light-chain kinase in endothelial cell retraction

    SciTech Connect

    Wysolmerski, R.B.; Lagunoff, D. )

    1990-01-01

    Permeabilized bovine pulmonary artery endothelial cell monolayers were used to investigate the mechanism of endothelial cell retraction. Postconfluent endothelial cells permeabilized with saponin retracted upon exposure to ATP and Ca{sup 2+}. Retraction was accompanied by thiophosphorylation of 19,000-Da myosin light chains when adenosine 5'-(gamma-({sup 35}S)thio)triphosphate was included in the medium. Both retraction and thiophosphorylation of myosin light chains exhibited a graded quantitative dependence on Ca{sup 2+}. When permeabilized monolayers were extracted in buffer D containing 100 mM KCl and 30 mM MgCl2 for 30 min, the cells failed to retract upon exposure to ATP and Ca{sup 2+}, and no thiophosphorylation of myosin light chains occurred. The ability both to retract and to thiophosphorylate myosin light chains was restored by the addition to the permeabilized, extracted cells of myosin light-chain kinase and calmodulin together but not by either alone. These studies indicate that endothelial cell retraction, as does smooth muscle contraction, depends on myosin light-chain kinase phosphorylation of myosin light chains.

  9. Antigen nature and complexity influence human antibody light chain usage and specificity.

    PubMed

    Smith, Kenneth; Shah, Hemangi; Muther, Jennifer J; Duke, Angie L; Haley, Kathleen; James, Judith A

    2016-05-27

    Human antibodies consist of a heavy chain and one of two possible light chains, kappa (κ) or lambda (λ). Here we tested how these two possible light chains influence the overall antibody response to polysaccharide and protein antigens by measuring light chain usage in human monoclonal antibodies from antibody secreting cells obtained following vaccination with Pneumovax23. Remarkably, we found that individuals displayed restricted light chain usage to certain serotypes and that lambda antibodies have different specificities and modes of cross-reactivity than kappa antibodies. Thus, at both the monoclonal (7 kappa, no lambda) and serum levels (145μg/mL kappa, 2.82μg/mL lambda), antibodies to cell wall polysaccharide were nearly always kappa. The pneumococcal reference serum 007sp was analyzed for light chain usage to 12 pneumococcal serotypes for which it is well characterized. Similar to results at the monoclonal level, certain serotypes tended to favor one of the light chains (14 and 19A, lambda; 6A and 23F, kappa). We also explored differences in light chain usage at the serum level to a variety of antigens. We examined serum antibodies to diphtheria toxin mutant CRM197 and Epstein-Barr virus protein EBNA-1. These responses tended to be kappa dominant (average kappa-to-lambda ratios of 4.52 and 9.72 respectively). Responses to the influenza vaccine were more balanced with kappa-to-lambda ratio averages having slight strain variations: seasonal H1N1, 1.1; H3N2, 0.96; B, 0.91. We conclude that antigens with limited epitopes tend to produce antibodies with restricted light chain usage and that in most individuals, antibodies with lambda light chains have specificities different and complementary to kappa-containing antibodies.

  10. Antigen nature and complexity influence human antibody light chain usage and specificity.

    PubMed

    Smith, Kenneth; Shah, Hemangi; Muther, Jennifer J; Duke, Angie L; Haley, Kathleen; James, Judith A

    2016-05-27

    Human antibodies consist of a heavy chain and one of two possible light chains, kappa (κ) or lambda (λ). Here we tested how these two possible light chains influence the overall antibody response to polysaccharide and protein antigens by measuring light chain usage in human monoclonal antibodies from antibody secreting cells obtained following vaccination with Pneumovax23. Remarkably, we found that individuals displayed restricted light chain usage to certain serotypes and that lambda antibodies have different specificities and modes of cross-reactivity than kappa antibodies. Thus, at both the monoclonal (7 kappa, no lambda) and serum levels (145μg/mL kappa, 2.82μg/mL lambda), antibodies to cell wall polysaccharide were nearly always kappa. The pneumococcal reference serum 007sp was analyzed for light chain usage to 12 pneumococcal serotypes for which it is well characterized. Similar to results at the monoclonal level, certain serotypes tended to favor one of the light chains (14 and 19A, lambda; 6A and 23F, kappa). We also explored differences in light chain usage at the serum level to a variety of antigens. We examined serum antibodies to diphtheria toxin mutant CRM197 and Epstein-Barr virus protein EBNA-1. These responses tended to be kappa dominant (average kappa-to-lambda ratios of 4.52 and 9.72 respectively). Responses to the influenza vaccine were more balanced with kappa-to-lambda ratio averages having slight strain variations: seasonal H1N1, 1.1; H3N2, 0.96; B, 0.91. We conclude that antigens with limited epitopes tend to produce antibodies with restricted light chain usage and that in most individuals, antibodies with lambda light chains have specificities different and complementary to kappa-containing antibodies. PMID:27113164

  11. Transfection of an immunoglobulin kappa gene into mature human B lymphocytes

    SciTech Connect

    Bich-Thuy, L.T.; Queen, C.

    1988-01-01

    The authors show in this report that the transcription induced by interleukin-2 or pokeweed mitogens of the kappa MOPC 41 immunoglobulin light-chain gene transfected into primary human or murine B lymphocytes initiates from a previously unobserved start site about 26 base pairs upstream of the start site used in myeloma cell lines.

  12. Clathrin light chains: arrays of protein motifs that regulate coated-vesicle dynamics.

    PubMed

    Brodsky, F M; Hill, B L; Acton, S L; Näthke, I; Wong, D H; Ponnambalam, S; Parham, P

    1991-06-01

    Polymerization of clathrin triskelions into clathrin coats and subsequent disassembly by the heat shock protein hsc70 control receptor-mediated pathways of intracellular transport. The clathrin light chains are major regulatory elements in these processes. These polypeptides consist of linear arrays of functional domains with distinctive sequence motifs. Comparison of unicellular and multicellular eukaryotes reveals differences in the numbers of clathrin light chains and in the functional domains they contain. PMID:1909824

  13. Light Chain Escape in 3 Cases: Evidence of Intraclonal Heterogeneity in Multiple Myeloma from a Single Institution in Poland.

    PubMed

    Kraj, Maria; Kruk, Barbara; Endean, Kelly; Warzocha, Krzysztof; Budziszewska, Katarzyna; Dąbrowska, Monika

    2015-01-01

    We report three cases of light chain escape (LCE) at a single institution in Poland, including an interesting case of biclonal monoclonal gammopathy of undetermined significance (MGUS) that satisfied the criteria for progression to light chain multiple myeloma (LCMM) with a rapid rise in serum free light chain (FLC) levels, following steroidal treatment for simultaneous temporal artery inflammation and polymyalgia rheumatica (PMR). In the three cases discussed, progression of the disease by light chain escape was associated with rapid and severe renal impairment, highlighting the necessity for prompt detection of such free light chain-only producing clones in order to prevent the possible development of irreversible end-organ damage. Interestingly, monitoring of these three patients by serum free light chain assay (sFLC) and retrospective heavy/light chain analysis (HLC) detected this clonal evolution prior to clinical relapse and suggests that these assays represent important additional tools for more accurate monitoring of multiple myeloma patients. PMID:26881153

  14. Light Chain Escape in 3 Cases: Evidence of Intraclonal Heterogeneity in Multiple Myeloma from a Single Institution in Poland

    PubMed Central

    Kraj, Maria; Kruk, Barbara; Endean, Kelly; Warzocha, Krzysztof; Budziszewska, Katarzyna; Dąbrowska, Monika

    2015-01-01

    We report three cases of light chain escape (LCE) at a single institution in Poland, including an interesting case of biclonal monoclonal gammopathy of undetermined significance (MGUS) that satisfied the criteria for progression to light chain multiple myeloma (LCMM) with a rapid rise in serum free light chain (FLC) levels, following steroidal treatment for simultaneous temporal artery inflammation and polymyalgia rheumatica (PMR). In the three cases discussed, progression of the disease by light chain escape was associated with rapid and severe renal impairment, highlighting the necessity for prompt detection of such free light chain-only producing clones in order to prevent the possible development of irreversible end-organ damage. Interestingly, monitoring of these three patients by serum free light chain assay (sFLC) and retrospective heavy/light chain analysis (HLC) detected this clonal evolution prior to clinical relapse and suggests that these assays represent important additional tools for more accurate monitoring of multiple myeloma patients. PMID:26881153

  15. Pseudo-Peritoneal Carcinomatosis Presentation of a Crystal-Storing Histiocytosis With an Unmutated Monoclonal κ Light Chain

    PubMed Central

    Aline-Fardin, Aude; Bender, Sebastien; Fabiani, Bettina; Buob, David; Brahimi, Said; Verpont, Marie Christine; Mothy, Mohamad; Ronco, Pierre; Boffa, Jean Jacques; Aucouturier, Pierre; Garderet, Laurent

    2015-01-01

    Abstract Crystal-storing histiocytosis (CSH) is a rare complication of monoclonal gammopathies caused by accumulation of crystalline material inside macrophages, and it may result in a variety of clinical manifestations depending on the involved organs. Although immunoglobulin κ light chains (LCs) seem to be the most frequent pathogenic component, very few molecular data are currently available. A 69-year-old man presented with a very poor performance status. Remarkable features were mesenteric lymph node enlargement and proteinuria, including a monoclonal κ LC. Light and electron microscopy studies revealed the presence of crystals within macrophages in the lymph nodes, bone marrow, and kidney, leading to the diagnosis of CSH. The pathogenic κ LC variable domain sequence was identical to the germline Vk3-20∗01/Jk2∗01 gene segments, without any somatic mutation, suggesting an extra-follicular B cell proliferation. The patient was successfully treated with 4 cycles of bortezomib and dexamethasone. After a 12-month follow-up, he remains in hematological and renal remission. CSH may present as pseudo-peritoneal carcinomatosis and relate to a monoclonal κ LC encoded by an unmutated gene. Bortezomib-based therapy proved efficacious in this case. PMID:26266355

  16. Pseudo-Peritoneal Carcinomatosis Presentation of a Crystal-Storing Histiocytosis With an Unmutated Monoclonal κ Light Chain.

    PubMed

    Aline-Fardin, Aude; Bender, Sebastien; Fabiani, Bettina; Buob, David; Brahimi, Said; Verpont, Marie Christine; Mothy, Mohamad; Ronco, Pierre; Boffa, Jean Jacques; Aucouturier, Pierre; Garderet, Laurent

    2015-08-01

    Crystal-storing histiocytosis (CSH) is a rare complication of monoclonal gammopathies caused by accumulation of crystalline material inside macrophages, and it may result in a variety of clinical manifestations depending on the involved organs. Although immunoglobulin κ light chains (LCs) seem to be the most frequent pathogenic component, very few molecular data are currently available.A 69-year-old man presented with a very poor performance status. Remarkable features were mesenteric lymph node enlargement and proteinuria, including a monoclonal κ LC. Light and electron microscopy studies revealed the presence of crystals within macrophages in the lymph nodes, bone marrow, and kidney, leading to the diagnosis of CSH. The pathogenic κ LC variable domain sequence was identical to the germline Vk3-2001/Jk201 gene segments, without any somatic mutation, suggesting an extra-follicular B cell proliferation.The patient was successfully treated with 4 cycles of bortezomib and dexamethasone. After a 12-month follow-up, he remains in hematological and renal remission.CSH may present as pseudo-peritoneal carcinomatosis and relate to a monoclonal κ LC encoded by an unmutated gene. Bortezomib-based therapy proved efficacious in this case.

  17. Characteristics of light chains of Chara myosin revealed by immunological investigation.

    PubMed

    Kakei, Toshihito; Sumiyoshi, Hiroki; Higashi-Fujime, Sugie

    2012-01-01

    Chara myosin is plant myosin responsible for cytoplasmic streaming and moves actin filaments at 60 µm/s, which is the fastest of all myosins examined. The neck of the myosin molecule has usually mechanical and regulatory roles. The neck of Chara myosin is supposed to bind six light chains, but, at present, we have no knowledge about them. We found Ca⁺⁺-calmodulin activated Chara myosin motility and its actin-activated ATPase, and actually bound with the Chara myosin heavy chain, indicating calmodulin might be one of candidates for Chara myosin light chains. Antibody against essential light chain from Physarum myosin, and antibodies against Chara calmodulin and chicken myosin light chain from lens membranes reacted with 20 kDa and 18 kDa polypeptides of Chara myosin preparation, respectively. Correspondingly, column purified Chara myosin had light chains of 20 kDa, and 18 kDa with the molar ratio of 0.7 and 2.5 to the heavy chain, respectively.

  18. Minimum requirements for inhibition of smooth-muscle myosin light-chain kinase by synthetic peptides.

    PubMed Central

    Hunt, J T; Floyd, D M; Lee, V G; Little, D K; Moreland, S

    1989-01-01

    Although the amino acid residues that are important for peptide substrates of myosin light-chain kinase have been reported, those that are important for peptide inhibitors of this enzyme have not previously been investigated. Synthetic peptides based on the sequence Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -Asn-Val21-Phe22-Ala of the chicken gizzard myosin light chain were tested as inhibitors of pig carotid-artery myosin light-chain kinase. The basic amino acid residues of the known myosin light-chain kinase inhibitor Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser-NH2 (IC50 = 14 microM) [Pearson, Misconi & Kemp (1986) J. Biol. Chem. 261, 25-27] were shown to be the important residues that contribute to inhibitor potency, as evidence by the finding that the hexapeptide Lys-Lys-Arg-Ala-Ala-Arg-NH2 had an IC50 value of 22 microM. This indicates that binding of the phosphorylatable serine residue to myosin light-chain kinase, which is of obvious importance for a substrate, does not enhance the potency of an inhibitor. With the aim of preparing more potent inhibitors, peptides Lys-Lys-Arg-Ala-Ala-Arg-Ala-Ala-Xaa-NH2 were prepared with a variety of amino acids substituted for the phosphorylatable serine residue. None of these peptides was a more potent inhibitor than the serine peptide. PMID:2920029

  19. Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein.

    PubMed

    Timofeeva, Anna M; Buneva, Valentina N; Nevinsky, Georgy A

    2015-10-01

    Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis.

  20. Structural basis of light chain amyloidogenicity: comparison of the thermodynamic properties, fibrillogenic potential and tertiary structural features of four Vlambda6 proteins.

    PubMed

    Wall, Jonathan S; Gupta, Vibha; Wilkerson, Matthew; Schell, Maria; Loris, Remy; Adams, Paul; Solomon, Alan; Stevens, Fred; Dealwis, Chris

    2004-01-01

    Primary (AL) amyloidosis results from the pathologic deposition of monoclonal light chains as amyloid fibrils. Studies of recombinant-derived variable region (VL) fragments of these proteins have shown an inverse relationship between thermodynamic stability and fibrillogenic potential. Further, ionic interactions within the VL domain were predicted to influence the kinetics of light chain fibrillogenicity, as evidenced from our analyses of a relatively stable Vlambda6 protein (Jto) with a long range electrostatic interaction between Asp and Arg side chains at position 29 and 68, respectively, and an unstable, highly fibrillogenic Vlambda6 protein (Wil) that had neutral amino acids at these locations. To test this hypothesis, we have generated two Jto-related mutants designed to disrupt the interaction between Asp 29 and Arg 68 (JtoD29A and JtoR68S). Although the thermodynamic stabilities of unfolding for these two molecules were identical, they exhibited very different kinetics of fibril formation: the rate of JtoD29A fibrillogenesis was slow and comparable to the parent molecule, whereas that of JtoR68S was significantly faster. High-resolution X-ray diffraction analyses of crystals prepared from the two mutants having the same space group and unit cell dimensions revealed no significant main-chain conformational changes. However, several notable side-chain alterations were observed in JtoR68S, as compared with JtoD29A, that resulted in the solvent exposure of a greater hydrophobic surface and modifications in the electrostatic potential surface. We posit that these differences contributed to the enhanced fibrillogenic potential of the Arg 68 mutant, since both Jto mutants lacked the intrachain ionic interaction and were equivalently unstable. The information gleaned from our studies has provided insight into structural parameters that in addition to overall thermodynamic stability, contribute to the fibril forming propensity of immunoglobulin light chains.

  1. Structural basis of light chain amyloidogenicity: comparison of the thermodynamic properties, fibrillogenic potential and tertiary structural features of four vλ6 proteins

    SciTech Connect

    Wall, J.S.; Gupta, V.; Wilkerson, M.; Schell, M.; Loris, R.; Adams, P.; Solomon, A.; Stevens, F.; Dealwis, C.

    2004-04-01

    immunoglobulin light chains.

  2. Bruton's Tyrosine Kinase Regulates the Activation of Gene Rearrangements at the λ Light Chain Locus in Precursor B Cells in the Mouse

    PubMed Central

    Dingjan, Gemma M.; Middendorp, Sabine; Dahlenborg, Katarina; Maas, Alex; Grosveld, Frank; Hendriks, Rudolf W.

    2001-01-01

    Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase involved in precursor B (pre-B) cell receptor signaling. Here we demonstrate that Btk-deficient mice have an ∼50% reduction in the frequency of immunoglobulin (Ig) λ light chain expression, already at the immature B cell stage in the bone marrow. Conversely, transgenic mice expressing the activated mutant BtkE41K showed increased λ usage. As the κ/λ ratio is dependent on (a) the level and kinetics of κ and λ locus activation, (b) the life span of pre-B cells, and (c) the extent of receptor editing, we analyzed the role of Btk in these processes. Enforced expression of the Bcl-2 apoptosis inhibitor did not alter the Btk dependence of λ usage. Crossing 3-83μδ autoantibody transgenic mice into Btk-deficient mice showed that Btk is not essential for receptor editing. Also, Btk-deficient surface Ig+ B cells that were generated in vitro in interleukin 7-driven bone marrow cultures manifested reduced λ usage. An intrinsic defect in λ locus recombination was further supported by the finding in Btk-deficient mice of reduced λ usage in the fraction of pre-B cells that express light chains in their cytoplasm. These results implicate Btk in the regulation of the activation of the λ locus for V(D)J recombination in pre-B cells. PMID:11369788

  3. Formation of assemblies on cell membranes by secreted proteins: molecular studies of free λ light chain aggregates found on the surface of myeloma cells.

    PubMed

    Hutchinson, Andrew T; Malik, Ansha; Berkahn, Mark B; Agostino, Mark; To, Joyce; Tacchi, Jessica L; Djordjevic, Steven P; Turnbull, Lynne; Whitchurch, Cynthia B; Edmundson, Allen B; Jones, Darren R; Raison, Robert L; Ramsland, Paul A

    2013-09-15

    We have described the presence of cell-membrane-associated κFLCs (free immunoglobulin light chains) on the surface of myeloma cells. Notably, the anti-κFLC mAb (monoclonal antibody) MDX-1097 is being assessed in clinical trials as a therapy for κ light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known whether λFLCs can associate with cell membranes of myeloma cells. In the present paper, we describe the presence of λFLCs on the surface of myeloma cells. We found that cell-surface-associated λFLCs are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that λFLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated λFLCs are aggregated. Given the present findings, we propose a model whereby individual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen.

  4. Evaluation of serum markers in the LRF CLL4 trial: β2-microglobulin but not serum free light chains, is an independent marker of overall survival.

    PubMed

    Pratt, Guy; Thomas, Peter; Marden, Nicola; Alexander, Denis; Davis, Zadie; Hussey, David; Parry, Helen; Harding, Stephen; Catovsky, Daniel; Begley, Joe; Oscier, David

    2016-10-01

    Chronic lymphocytic leukemia (CLL) is characterized by heterogeneous clinical behavior and there is a need for improved biomarkers. The current study evaluated the prognostic significance of serum free light chains (sFLC, kappa, and lambda) and other serum markers (bar, serum thymidine kinase (sTK), soluble CD23, and LDH) together with established biomarkers in 289 patients enrolled into the LRF CLL4 trial. In a multivariable analysis of serum markers alone, higher big and kappa light chains were statistically significant in predicting disease progression and higher blg, and sTK in predicting mortality. In multivariable analysis for overall survival the following were independently significant: β2M levels, immunoglobulin gene (IGHV) mutational status (>98% homology), age, 17p13 deletions (>10%), and CD38 expression. β2M is the only serum marker that retained clear independent value as a biomarker in the LRF CLL4 trial and remains powerfully prognostic requiring evaluation in any future method of risk stratifying patients.

  5. Regulatory and essential light chains of myosin rotate equally during contraction of skeletal muscle.

    PubMed

    Borejdo, Julian; Ushakov, Dmitry S; Akopova, Irina

    2002-06-01

    Myosin head consists of a globular catalytic domain and a long alpha-helical regulatory domain. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. The proximal end of the regulatory domain contains the essential light chain 1 (LC1). This light chain can interact through the N and C termini with actin and myosin heavy chain. The interactions may inhibit the motion of the proximal end. In consequence the motion of the distal end (containing regulatory light chain, RLC) may be different from the motion of the proximal end. To test this possibility, the angular motion of LC1 and RLC was measured simultaneously during muscle contraction. Engineered LC1 and RLC were labeled with red and green fluorescent probes, respectively, and exchanged with native light chains of striated muscle. The confocal microscope was modified to measure the anisotropy from 0.3 microm(3) volume containing approximately 600 fluorescent cross-bridges. Static measurements revealed that the magnitude of the angular change associated with transition from rigor to relaxation was less than 5 degrees for both light chains. Cross-bridges were activated by a precise delivery of ATP from a caged precursor. The time course of the angular change consisted of a fast phase followed by a slow phase and was the same for both light chains. These results suggest that the interactions of LC1 do not inhibit the angular motion of the proximal end of the regulatory domain and that the whole domain rotates as a rigid body.

  6. Significant Differences in Physicochemical Properties of Human Immunoglobulin Kappa and Lambda CDR3 Regions

    PubMed Central

    Townsend, Catherine L.; Laffy, Julie M. J.; Wu, Yu-Chang Bryan; Silva O’Hare, Joselli; Martin, Victoria; Kipling, David; Fraternali, Franca; Dunn-Walters, Deborah K.

    2016-01-01

    Antibody variable regions are composed of a heavy and a light chain, and in humans, there are two light chain isotypes: kappa and lambda. Despite their importance in receptor editing, the light chain is often overlooked in the antibody literature, with the focus being on the heavy chain complementarity-determining region (CDR)-H3 region. In this paper, we set out to investigate the physicochemical and structural differences between human kappa and lambda light chain CDR regions. We constructed a dataset containing over 29,000 light chain variable region sequences from IgM-transcribing, newly formed B cells isolated from human bone marrow and peripheral blood. We also used a published human naïve dataset to investigate the CDR-H3 properties of heavy chains paired with kappa and lambda light chains and probed the Protein Data Bank to investigate the structural differences between kappa and lambda antibody CDR regions. We found that kappa and lambda light chains have very different CDR physicochemical and structural properties, whereas the heavy chains with which they are paired do not differ significantly. We also observed that the mean CDR3 N nucleotide addition in the kappa, lambda, and heavy chain gene rearrangements are correlated within donors but can differ between donors. This indicates that terminal deoxynucleotidyl transferase may work with differing efficiencies between different people but the same efficiency in the different classes of immunoglobulin chain within one person. We have observed large differences in the physicochemical and structural properties of kappa and lambda light chain CDR regions. This may reflect different roles in the humoral immune response. PMID:27729912

  7. Purification, Characterization and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkia.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in close species. In this study, MLC with the molecular mass of 18kDa was purified from crayfish (Procambarus clarkii) muscle fibrils. Its physicochemical characterization showed that the...

  8. Expression of immunoglobulin kappa and lambda chains in mink.

    PubMed

    Bovkun, L A; Peremislov, V V; Nayakshin, A M; Belousov, E S; Mechetina, L V; Aasted, B; Taranin, A V

    1993-08-01

    The ratio of kappa and lambda chains of immunoglobulins varies significantly from one species to another. It has previously been thought that lambda was only type expressed in mink. We tested mink immunoglobulin light chains using two monoclonal antibodies G80 and G88. It has been shown that G80 and G88 specifically recognize two antigenically different subpopulations of the light chains. Immunochemical analysis of these subpopulations separated by affinity chromatography suggested that they represent lambda and kappa types of light chains, respectively. Screening of a mink cDNA library with monoclonal antibody G88 resulted in the isolation of clone pIGK-1 containing kappa chain-encoding sequence. The cDNA insert of pIGK-1 included most of the V segment, as well as the J, C and 3' untranslated sequences. Mink V kappa sequence shown the highest homology with the human V kappa II subgroup genes (76-79%). Mink C kappa sequence was 53-63% homologous to C kappa of other species. The striking feature of mink C kappa chain is the presence of glutamine in the C-terminal position. Southern blot analysis suggested that mink haploid genome has one C kappa gene and multiple V kappa genes. The kappa:lambda chain ratio in the 12 minks studied was, on the average, 46:54. The same ratio was observed for the kappa- and lambda-producing cells in the mesenteric lymph nodes. The five previously identified mink light chain allotypes were assigned to the lambda chains, thereby confirming that lambda chains in this species are additionally subdivided into several subtypes.

  9. Omega Electroproduction

    SciTech Connect

    Unwuchola, A. D.; Connell, Simon H.; Aurousseau, M.; Dalton, Mark M.

    2013-08-01

    The differential cross section for p(e, e'ω)p has been studied at Q{sup 2} ~ 5.5 (GeV/c)2. Here Q{sup 2} represents the four momentum squared of the virtual photon in the excitation of baryonic resonances by an electron projectile. In order to extract the ω-meson differential cross section from the JLAB data, the data was compared to a full Monte Carlo simulation of the detector based on events generated for omega production in a way that the production cross section was varied to achieve a match to the data. The bin selected for this procedure takes into account the measure of robustness of the stripping of the ω peak from the multi-pion background as well as the statistics in the measured data and the Monte Carlo simulation of the signal and background physics. An error estimation technique for the cross section was based on determining the dependence of the extracted cross section parameters on the experimental set-up (including parameters for the spectrometer, target beam geometeries and performance). We compare our results with a Regge-based model for hadronic content in the t-channel exchange of a photon in Q{sup 2} region of overlap. There is an extension of this data into a completely new region, which is the highest yet measured.

  10. Structure of the light chain-binding domain of myosin V

    PubMed Central

    Terrak, Mohammed; Rebowski, Grzegorz; Lu, Renne C.; Grabarek, Zenon; Dominguez, Roberto

    2005-01-01

    Myosin V is a double-headed molecular motor involved in organelle transport. Two distinctive features of this motor, processivity and the ability to take extended linear steps of ≈36 nm along the actin helical track, depend on its unusually long light chain-binding domain (LCBD). The LCBD of myosin V consists of six tandem IQ motifs, which constitute the binding sites for calmodulin (CaM) and CaM-like light chains. Here, we report the 2-Å resolution crystal structure of myosin light chain 1 (Mlc1p) bound to the IQ2–IQ3 fragment of Myo2p, a myosin V from Saccharomyces cerevisiae. This structure, combined with FRET distance measurements between probes in various CaM–IQ complexes, comparative sequence analysis, and the previously determined structures of Mlc1p-IQ2 and Mlc1p-IQ4, allowed building a model of the LCBD of myosin V. The IQs of myosin V are distributed into three pairs. There appear to be specific cooperative interactions between light chains within each IQ pair, but little or no interaction between pairs, providing flexibility at their junctions. The second and third IQ pairs each present a light chain, whether CaM or a CaM-related molecule, bound in a noncanonical extended conformation in which the N-lobe does not interact with the IQ motif. The resulting free N-lobes may engage in protein–protein interactions. The extended conformation is characteristic of the single IQ of myosin VI and is common throughout the myosin superfamily. The model points to a prominent role of the LCBD in the function, regulation, and molecular interactions of myosin V. PMID:16120677

  11. Unusual intracytoplasmic immunoglobulin inclusions in chronic lymphocytic leukaemia.

    PubMed

    Guglielmi, P; Preud'Homme, J L; Gourdin, M F; Reyes, F; Daniel, M T

    1982-01-01

    Unusual intracytoplasmic immunoglobulin inclusions were found by immunofluorescence in three patients with chronic lymphocytic leukaemia. The inclusions contained the same immunoglobulin chains as those detected on the plasma membrane, except for delta chains which were expressed on the cell surface and not in the cytoplasmic inclusions. The cytoplasmic staining persisted throughout culture for 8 or more days. An initial study of patients 1's cells showed that the inclusions contained only mu chains, and kappa chains gradually became apparent after in vitro culture. In a second study, the fresh lymphocytes contained both mu and and kappa chains. Initially, biosynthetic experiments showed production of mu chains which polymerized in the cytoplasm and were not secreted. Subsequently there was synthesis of heavy and light chains which assembled into monomeric subunits that were retained and secretion of free light chains. The apparent molecular weight of these immunoglobulin chains was larger than that of their secretory counterparts. Immunoelectronmicroscopy revealed cytoplasmic mu chains in strands of endoplasmic reticulum. In the two other patients, immunofluorescence displayed unusual staining patterns of bright networks in perinuclear areas. PMID:6275878

  12. Organization and expression of immunoglobulin genes in fetal liver hybridomas.

    PubMed

    Perry, R P; Kelley, D E; Coleclough, C; Kearney, J F

    1981-01-01

    The organization and expression of immunoglobulin genes were studied in a series of six hybridomas derived from the fusion of a nonproducing myeloma cell with cells from mouse fetal liver. These hybridomas, which exhibit several phenotypic characteristics of immature B lymphocytes, all have productively rearranged mu heavy chain genes and produce both the membrane and secreted forms of mu mRNA in a ratio of about 1:10. Significantly, none of the hybridomas has an unrearranged (germ line) allelic mu gene. Examination of the kappa light chain genes revealed that all six of the hybridomas contain unrearranged kappa loci and produce 8.4-kilobase transcripts containing kappa constant region sequences. None of the five hybridomas that exhibit a mu-only phenotype contains a rearranged kappa gene other than that derived from the myeloma parent. One hybridoma, which actively secretes kappa immunoglobulin, contains a rearranged kappa gene of fetal liver origin and synthesizes a distinctive kappa mRNA precursor in addition to the 8.4-kilobase transcript. These results demonstrate that rearrangement of heavy chain immunoglobulin genes normally occurs prior to that of light chain genes and further indicate that the transcriptional competence of the kappa constant region locus is established prior to the time of its rearrangement.

  13. Resonances in the {omega}{omega} system

    SciTech Connect

    Amelin, D. V.; Bugg, D. V.; Gavrilov, Yu. G.; Gouz, Yu. P.; Dzhelyadin, R. I.; Dorofeev, V. A.; Zaitsev, A. M.; Zenin, A. V.; Ivashin, A. V.; Kabachenko, V. V.; Karyukhin, A. N.; Kachaev, I. A.; Konoplyannikov, A. N.; Konstantinov, V. F.; Kostyukhin, V. V.; Matveev, V. D.; Nikolaenko, V. I.; Ostankov, A. P.; Polyakov, B. F.; Ryabchikov, D. I.

    2006-04-15

    Results of a partial-wave analysis for the reaction {pi}{sup -} {sup {yields}} {omega}{omega}n studied at the VES spectrometer (Institute for High Energy Physics, Protvino) are presented. The behavior of the J{sup PC} = 2{sup ++} amplitudes in the {omega}{omega} system is described by the f{sub 2}(1565) and f{sub 2}(1910) resonances, whose parameters were fixed at M = 1.590 {+-} 0.010 GeV and {gamma} = 0.140 {+-} 0.011 GeV for the former and at M = 1.890 {+-} 0.010 GeV and {gamma} = 0.165 {+-} 0.019 GeV for the latter. The decay f{sub 4}(2050) {sup {yields}} {omega}{omega} was observed at parameters values of M = 1.960 {+-} 0.015 GeV, {gamma} = 0.290 {+-} 0.020 GeV.

  14. Omega documentation

    SciTech Connect

    Howerton, R.J.; Dye, R.E.; Giles, P.C.; Kimlinger, J.R.; Perkins, S.T.; Plechaty, E.F.

    1983-08-01

    OMEGA is a CRAY I computer program that controls nine codes used by LLNL Physical Data Group for: 1) updating the libraries of evaluated data maintained by the group (UPDATE); 2) calculating average values of energy deposited in secondary particles and residual nuclei (ENDEP); 3) checking the libraries for internal consistency, especially for energy conservation (GAMCHK); 4) producing listings, indexes and plots of the library data (UTILITY); 5) producing calculational constants such as group averaged cross sections and transfer matrices for diffusion and Sn transport codes (CLYDE); 6) producing and updating standard files of the calculational constants used by LLNL Sn and diffusion transport codes (NDFL); 7) producing calculational constants for Monte Carlo transport codes that use group-averaged cross sections and continuous energy for particles (CTART); 8) producing and updating standard files used by the LLNL Monte Carlo transport codes (TRTL); and 9) producing standard files used by the LANL pointwise Monte Carlo transport code MCNP (MCPOINT). The first four of these functions and codes deal with the libraries of evaluated data and the last five with various aspects of producing calculational constants for use by transport codes. In 1970 a series, called PD memos, of internal and informal memoranda was begun. These were intended to be circulated among the group for comment and then to provide documentation for later reference whenever questions arose about the subject matter of the memos. They have served this purpose and now will be drawn upon as source material for this more comprehensive report that deals with most of the matters covered in those memos.

  15. λ Light Chain Bias Associated With Enhanced Binding and Function of Anti-HIV Env Glycoprotein Antibodies.

    PubMed

    Sajadi, Mohammad M; Farshidpour, Maham; Brown, Eric P; Ouyang, Xin; Seaman, Michael S; Pazgier, Marzena; Ackerman, Margaret E; Robinson, Harriet; Tomaras, Georgia; Parsons, Matthew S; Charurat, Manhattan; DeVico, Anthony L; Redfield, Robert R; Lewis, George K

    2016-01-01

    The humoral response to human immunodeficiency virus (HIV) remains incompletely understood. In this report, we describe biased λ light chain use during the HIV Env glycoprotein (Env) response in HIV infection and vaccination. We examined HIV Env binding (and neutralization) in the context of light chain use in subjects with acute HIV infection, chronic HIV infection, and among HIV vaccinees. In all populations tested, there was a λ chain bias for HIV Env binding antibodies, compared with other HIV antigens (such as p24) or tetanus toxoid. In subjects with chronic HIV infection, a λ bias was noted for neutralization, with λ antibodies accounting for up to 90% of all neutralization activity observed. This is the first report of antibody function in a human infection being tied to light chain use. In HIV infection, antibodies expressing λ light chains tended to have longer CDRL3s, increased light chain contact with HIV Env, and less hypermutation in the heavy chain, compared with antibodies using the κ light chain. These data also support an evolutionary model for the understanding the various κ to λ light chain ratios observed across species and suggest that the λ light chain bias against HIV provides the host an advantage in developing a more efficient humoral response.

  16. Elevated PC responsive B cells and anti-PC antibody production in transgenic mice harboring anti-PC immunoglobulin genes.

    PubMed

    Pinkert, C A; Manz, J; Linton, P J; Klinman, N R; Storb, U

    1989-12-01

    The rearrangement of heavy and light chain immunoglobulin genes is necessary for the production of functional antibody molecules. The myeloma MOPC 167 produces specific antibodies to the antigen phosphorylcholine (PC), which is present on bacterial surfaces, fungi and other environmental contaminants. Rearranged heavy and light chain immunoglobulin genes cloned from MOPC 167 were microinjected into mouse eggs. Within the resulting transgenic mice, expression of the transgenes were limited to lymphoid tissues. Transgenic mice produced elevated levels of anti-PC antibodies constitutively, at 16 days of age, when normal non-transgenic mice were not fully immunocompetent. A triggering antigenic stimulus was not necessary to evoke anti-PC immunoglobulin production. Additionally, the frequency of PC-responsive B cells in these transgenic mice was further increased upon specific immunization.

  17. Identification of a DNA binding protein that recognizes the nonamer recombinational signal sequence of immunoglobulin genes.

    PubMed

    Halligan, B D; Desiderio, S V

    1987-10-01

    Extracts of nuclei from B- and T-lymphoid cells contain a protein that binds specifically to the conserved nonamer DNA sequence within the recombinational signals of immunoglobulin genes. Complexes with DNA fragments from four kappa light-chain joining (J) segments have the same electrophoretic mobility. Nonamer-containing DNA fragments from heavy-chain and light-chain genes compete for binding. Within the 5'-flanking DNA of the J kappa 4 gene segment, the binding site has been localized to a 27-base-pair interval spanning the nonamer region. The binding activity is recovered as a single peak after ion-exchange chromatography. The site of binding of the protein and its presence in nuclei of lymphoid cells suggest that it may function in the assembly of immunoglobulin genes.

  18. Light-chain deposition disease of the kidney: a case report.

    PubMed

    Darouich, Sihem; Goucha, Rym; Jaafoura, Mohamed Habib; Zekri, Semy; Kheder, Adel; Maiz, Hedi Ben

    2012-04-01

    A 41-year-old man was admitted for evaluation of nephrotic syndrome associated with microhematuria, hypertension, and moderate renal failure. In serum and urine samples, monoclonal IgG-lambda was detected. Bone marrow examination showed normal representation of all cell lines with normal range of plasma cells. Renal biopsy demonstrated diabetes-like nodular glomerulosclerosis. Immunofluorescence failed to demonstrate the presence of kappa or lambda light chains in the kidney. Electron microcopy showed granular electron-dense deposits along the glomerular basement membranes and in the mesangial nodules. The patient was diagnosed as having light-chain deposition disease (LCDD) without evidence of plasma cell dyscrasia. This report was designed to stress the significant challenges that remain in the diagnosis of LCDD-related glomerulopathy. The salient morphological features that help in making an accurate diagnosis are discussed. PMID:22471437

  19. Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

    SciTech Connect

    Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.; Reshetnikova, L.; Brown, J. H.; Szent-Gyorgyi, A. G.; Cohen, C.

    2011-11-02

    We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

  20. Light-chain deposition disease of the kidney: a case report.

    PubMed

    Darouich, Sihem; Goucha, Rym; Jaafoura, Mohamed Habib; Zekri, Semy; Kheder, Adel; Maiz, Hedi Ben

    2012-04-01

    A 41-year-old man was admitted for evaluation of nephrotic syndrome associated with microhematuria, hypertension, and moderate renal failure. In serum and urine samples, monoclonal IgG-lambda was detected. Bone marrow examination showed normal representation of all cell lines with normal range of plasma cells. Renal biopsy demonstrated diabetes-like nodular glomerulosclerosis. Immunofluorescence failed to demonstrate the presence of kappa or lambda light chains in the kidney. Electron microcopy showed granular electron-dense deposits along the glomerular basement membranes and in the mesangial nodules. The patient was diagnosed as having light-chain deposition disease (LCDD) without evidence of plasma cell dyscrasia. This report was designed to stress the significant challenges that remain in the diagnosis of LCDD-related glomerulopathy. The salient morphological features that help in making an accurate diagnosis are discussed.

  1. Omega-6 Fatty Acids

    MedlinePlus

    ... types of fats. Some types are found in vegetable oils, including corn, evening primrose seed, safflower, and soybean ... from studying specific omega-6 fatty acids or plant oils containing omega-6 fatty acids. See the separate ...

  2. EPR and CD spectroscopy of fast myosin light chain conformation during binding of trifluoperazine.

    PubMed

    Huang, W; Wilson, G J; Brown, L J; Lam, H; Hambly, B D

    1998-10-15

    The conformations of isolated rabbit fast myosin light chains (LCs) were modified using trifluoperazine (TFP), the hydrophobic calmodulin inhibitor. CD spectroscopy showed that TFP altered secondary structural content of the LCs, with half-maximal effects at TFP concentrations of approximately 14-50 microM, which is within the range required to alter muscle fiber contraction in both agonistic and antagonistic ways [Kurebayashi, N. & Ogawa, Y. (1988) J. Physiol. 403, 407-424]. EPR spectroscopy provided structural information from paramagnetic probes on C-terminal domain surfaces. In the absence of TFP, tauR (rotational correlation time) was 1.6 ns for both alkali light chains (ALCs) and 1.8 ns for light chain 2 (LC2). This was faster than expected for proteins of this size (approximately 10 ns). TFP progressively recruited the probes into populations with tauR sevenfold to 12-fold slower, with half-maximal effects at a TFP concentration of approximately 370-800 microM. The differences probably indicate that CD spectroscopy detects changes in protein conformation due to 'specific' TFP binding at the LC hydrophobic core, while less specific binding at higher TFP concentrations is required to effect conformational changes on the protein surfaces near the paramagnetic probes. TFP binding was generally not cooperative. Comparative sequence analysis between calmodulin, troponin C, and myosin LCs indicated considerable conservation between residues expected to bind TFP.

  3. What is new in diagnosis and management of light chain amyloidosis?

    PubMed

    Palladini, Giovanni; Merlini, Giampaolo

    2016-07-14

    Light chain (AL) amyloidosis is caused by a usually small plasma cell clone producing a misfolded light chain that deposits in tissues. Survival is mostly determined by the severity of heart involvement. Recent studies are clarifying the mechanisms of cardiac damage, pointing to a toxic effect of amyloidogenic light chains and offering new potential therapeutic targets. The diagnosis requires adequate technology, available at referral centers, for amyloid typing. Late diagnosis results in approximately 30% of patients presenting with advanced, irreversible organ involvement and dying in a few months despite modern treatments. The availability of accurate biomarkers of clonal and organ disease is reshaping the approach to patients with AL amyloidosis. Screening of early organ damage based on biomarkers can help identify patients with monoclonal gammopathy of undetermined significance who are developing AL amyloidosis before they become symptomatic. Staging systems and response assessment based on biomarkers facilitate the design and conduction of clinical trials, guide the therapeutic strategy, and allow the timely identification of refractory patients to be switched to rescue therapy. Treatment should be risk-adapted. Recent studies are linking specific characteristics of the plasma cell clone to response to different types of treatment, moving toward patient-tailored therapy. In addition, novel anti-amyloid treatments are being developed that might be combined with anti-plasma cell chemotherapy. PMID:27053535

  4. Tumor Stiffness Is Unrelated to Myosin Light Chain Phosphorylation in Cancer Cells

    PubMed Central

    Fry, Madeline; Greene, Madelyne; Chernaya, Olga; Hu, Wen-Yang; Chew, Teng-Leong; Mahmud, Nadim; Kadkol, Shrihari S.; Glover, Sarah; Prins, Gail; Strakova, Zuzana; de Lanerolle, Primal

    2013-01-01

    Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors. PMID:24224004

  5. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis

    PubMed Central

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G.; González-Reyes, Acaimo; Martín-Bermudo, María D.

    2016-01-01

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies. PMID:26888436

  6. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis.

    PubMed

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G; González-Reyes, Acaimo; Martín-Bermudo, María D

    2016-02-18

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies.

  7. Analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire.

    PubMed

    de Wildt, R M; Hoet, R M; van Venrooij, W J; Tomlinson, I M; Winter, G

    1999-01-22

    In the bone marrow, diversity in the primary antibody repertoire is created by the combinatorial rearrangement of different gene segments and by the association of different heavy and light chains. During the secondary response in the germinal centres, antibodies are diversified by somatic mutation and possibly by further rearrangements, or "receptor editing". Here, we have analysed the pairings of heavy and light chain variable domains (VH and VL) in 365 human IgG+ B cells from peripheral blood, and established that these pairings are largely random. The repertoire is dominated by a limited number of pairings of segments and folds. Among these pairings we identified two identical mutated heavy chains in combination with two different mutated light chains (one kappa and one lambda). This shows that receptor editing occurs in the human periphery and that the same antibody lineage can be subjected to both receptor editing and somatic hypermutation. This suggests that receptor editing may be used together with somatic mutation for the affinity maturation of antibodies. We also propose that receptor editing has shaped variable gene segment use and the evolution of V gene families.

  8. Glomerular disease with idiopathic linear immunoglobulin deposition: a rose by any other name would be atypical.

    PubMed

    Rosales, Ivy A; Colvin, Robert B

    2016-04-01

    Nasr et al. (2016) report 20 cases of "atypical anti-GBM disease," characterized by bright, linear glomerular basement membrane (GBM) immunoglobulin deposition in patients who lacked anti-GBM antibodies by conventional testing and who had a relatively benign course. Half had light chain restriction. The term "idiopathic linear immunoglobulin deposition" may be preferable until anti-GBM activity is demonstrable, since there are alternative mechanisms of linear deposition in the GBM, including physicochemical affinity for GBM components and alterations of the GBM itself.

  9. Cloning and sequencing of human lambda immunoglobulin genes by the polymerase chain reaction.

    PubMed

    Songsivilai, S; Bye, J M; Marks, J D; Hughes-Jones, N C

    1990-12-01

    Universal oligonucleotide primers, designed for amplifying and sequencing genes encoding the rearranged human lambda immunoglobulin variable region, were validated by amplification of the lambda light chain genes from four human heterohybridoma cell lines and in the generation of a cDNA library of human V lambda sequences from Epstein-Barr virus-transformed human peripheral blood lymphocytes. This technique allows rapid cloning and sequencing of human immunoglobulin genes, and has potential applications in the rescue of unstable human antibody-producing cell lines and in the production of human monoclonal antibodies.

  10. Thirteen is enough: the myosins of Dictyostelium discoideum and their light chains

    PubMed Central

    Kollmar, Martin

    2006-01-01

    Background Dictyostelium discoideum is one of the most famous model organisms for studying motile processes like cell movement, organelle transport, cytokinesis, and endocytosis. Members of the myosin superfamily, that move on actin filaments and power many of these tasks, are tripartite proteins consisting of a conserved catalytic domain followed by the neck region consisting of a different number of so-called IQ motifs for binding of light chains. The tails contain functional motifs that are responsible for the accomplishment of the different tasks in the cell. Unicellular organisms like yeasts contain three to five myosins while vertebrates express over 40 different myosin genes. Recently, the question has been raised how many myosins a simple multicellular organism like Dictyostelium would need to accomplish all the different motility-related tasks. Results The analysis of the Dictyostelium genome revealed thirteen myosins of which three have not been described before. The phylogenetic analysis of the motor domains of the new myosins placed Myo1F to the class-I myosins and Myo5A to the class-V myosins. The third new myosin, an orphan myosin, has been named MyoG. It contains an N-terminal extension of over 400 residues, and a tail consisting of four IQ motifs and two MyTH4/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) tandem domains that are separated by a long region containing an SH3 (src homology 3) domain. In contrast to previous analyses, an extensive comparison with 126 class-VII, class-X, class-XV, and class-XXII myosins now showed that MyoI does not group into any of these classes and should not be used as a model for class-VII myosins. The search for calmodulin related proteins revealed two further potential myosin light chains. One is a close homolog of the two EF-hand motifs containing MlcB, and the other, CBP14, phylogenetically groups to the ELC/RLC/calmodulin (essential light chain/regulatory light chain) branch of the tree

  11. Uniform detection of immunoglobulin-gene rearrangement in benign lymphoepithelial lesions.

    PubMed

    Fishleder, A; Tubbs, R; Hesse, B; Levine, H

    1987-04-30

    The term "benign lymphoepithelial lesion" is used to describe the salivary-gland lymphocytic infiltration and epithelial changes typically found in association with Sjögren's syndrome. We used Southern blot hybridization techniques to examine the immunoglobulin genes in salivary-gland tissue derived from eight patients with benign lymphoepithelial lesions. Three of these patients had intrasalivary non-Hodgkin's lymphoma complicating the lesions, whereas the lesions in the remaining five were all histologically benign. Ten samples from the eight patients all revealed rearrangement of both the heavy-chain and light-chain immunoglobulin genes. In one of the patients in whom non-Hodgkin's lymphoma involved both the salivary-gland lesion and an ipsilateral lymph node, the rearrangements of the heavy-chain and light-chain immunoglobulin genes detected at the two sites were identical. One other patient had two distinct benign lymphoepithelial lesions removed two years apart. The rearrangements of the heavy-chain as well as the kappa light-chain genes detected in these two lesions were entirely different. These data suggest that B-cell clonal expansion has an integral role in the pathophysiology of the benign lymphoepithelial lesion and may explain the increased incidence of lymphoma noted in association with this disorder.

  12. The immunoglobulins of cold-blooded vertebrates.

    PubMed

    Pettinello, Rita; Dooley, Helen

    2014-11-24

    Although lymphocyte-like cells secreting somatically-recombining receptors have been identified in the jawless fishes (hagfish and lamprey), the cartilaginous fishes (sharks, skates, rays and chimaera) are the most phylogenetically distant group relative to mammals in which bona fide immunoglobulins (Igs) have been found. Studies of the antibodies and humoral immune responses of cartilaginous fishes and other cold-blooded vertebrates (bony fishes, amphibians and reptiles) are not only revealing information about the emergence and roles of the different Ig heavy and light chain isotypes, but also the evolution of specialised adaptive features such as isotype switching, somatic hypermutation and affinity maturation. It is becoming increasingly apparent that while the adaptive immune response in these vertebrate lineages arose a long time ago, it is most definitely not primitive and has evolved to become complex and sophisticated. This review will summarise what is currently known about the immunoglobulins of cold-blooded vertebrates and highlight the differences, and commonalities, between these and more "conventional" mammalian species.

  13. The immunoglobulins of cold-blooded vertebrates.

    PubMed

    Pettinello, Rita; Dooley, Helen

    2014-01-01

    Although lymphocyte-like cells secreting somatically-recombining receptors have been identified in the jawless fishes (hagfish and lamprey), the cartilaginous fishes (sharks, skates, rays and chimaera) are the most phylogenetically distant group relative to mammals in which bona fide immunoglobulins (Igs) have been found. Studies of the antibodies and humoral immune responses of cartilaginous fishes and other cold-blooded vertebrates (bony fishes, amphibians and reptiles) are not only revealing information about the emergence and roles of the different Ig heavy and light chain isotypes, but also the evolution of specialised adaptive features such as isotype switching, somatic hypermutation and affinity maturation. It is becoming increasingly apparent that while the adaptive immune response in these vertebrate lineages arose a long time ago, it is most definitely not primitive and has evolved to become complex and sophisticated. This review will summarise what is currently known about the immunoglobulins of cold-blooded vertebrates and highlight the differences, and commonalities, between these and more "conventional" mammalian species. PMID:25427250

  14. The Immunoglobulins of Cold-Blooded Vertebrates

    PubMed Central

    Pettinello, Rita; Dooley, Helen

    2014-01-01

    Although lymphocyte-like cells secreting somatically-recombining receptors have been identified in the jawless fishes (hagfish and lamprey), the cartilaginous fishes (sharks, skates, rays and chimaera) are the most phylogenetically distant group relative to mammals in which bona fide immunoglobulins (Igs) have been found. Studies of the antibodies and humoral immune responses of cartilaginous fishes and other cold-blooded vertebrates (bony fishes, amphibians and reptiles) are not only revealing information about the emergence and roles of the different Ig heavy and light chain isotypes, but also the evolution of specialised adaptive features such as isotype switching, somatic hypermutation and affinity maturation. It is becoming increasingly apparent that while the adaptive immune response in these vertebrate lineages arose a long time ago, it is most definitely not primitive and has evolved to become complex and sophisticated. This review will summarise what is currently known about the immunoglobulins of cold-blooded vertebrates and highlight the differences, and commonalities, between these and more “conventional” mammalian species. PMID:25427250

  15. Immunoglobulin E in histoplasmosis.

    PubMed Central

    Cox, R A; Arnold, D R

    1980-01-01

    Immunoglobulin M, G, A, and E serum levels were quantitated in 20 patients with active histoplasmosis (group I), 24 healthy subjects who were skin test positive to histoplasmin (group II), and 47 healthy persons who were skin test negative to histoplasmin (group III). The results established that patients with this disease have increased immunoglobulin G (P less than 0.05), immunoglobulin A (P less than 0.001), and immunoglobulin E (P less than 0.01) serum levels when compared with the 71 healthy subjects in groups II and III. PMID:7399706

  16. Rapid cloning of any rearranged mouse immunoglobulin variable genes

    SciTech Connect

    Dattamajumdar, A.K.; Jacobson, D.P.; Hood, L.E.; Osman, G.E.

    1996-12-31

    Immunoglobulins (Ig) have been the focus of extensive study for several decades and have become an important research area for immunologists and molecular biologists. The use of polymerase chain reaction (PCR) technology has accelerated the cloning, sequencing, and characterization of genes of the immune system. However, cloning and sequencing the Ig variable (V) genes using the PCR technology has been a challenging task, primarily due to the very diverse nature of Ig V region genes. We have developed a simple, rapid, and reproducible PCR-based technique to clone any rearranged mouse Ig heavy or light chain genes. A close examination of all Ig heavy and light chain V gene families has resulted in the design of 5{prime} and 3{prime} universal primers from regions that are highly conserved across all heavy or light chain V gene families, and the joining or constant regions, respectively. We present our strategy for designing universal primers for Ig V gene families. These primers were able to rapidly amplify the rearranged Ig V genes, belonging to diverse Ig V gene families from very different cell lines, i.e., J558, MOPC-21, 36-60, and a chicken ovalbumin specific B-cell hybridoma. In addition, the present study provides the complete alignment of nucleotide sequences of all heavy and light chain variable gene families. This powerful method of cloning Ig V genes, therefore, allows rapid and precise analysis of B-cell hybridomas, B-cell repertoire, and B-cell ontogeny. 55 refs., 5 figs., 2 tabs.

  17. Clathrin light chain B: gene structure and neuron-specific splicing.

    PubMed Central

    Stamm, S; Casper, D; Dinsmore, J; Kaufmann, C A; Brosius, J; Helfman, D M

    1992-01-01

    The clathrin light chains are components of clathrin coated vesicles, structural constituents involved in endocytosis and membrane recycling. The clathrin light chain B (LCB) gene encodes two isoforms, termed LCB2 and LCB3, via an alternative RNA splicing mechanism. We have determined the structure of the rat clathrin light chain B gene. The gene consists of six exons that extend over 11.9 kb. The first four exons and the last exon are common to the LCB2 and LCB3 isoforms. The fifth exon, termed EN, is included in the mRNA in brain, giving rise to the brain specific form LCB2 but is excluded in other tissues, generating the LCB3 isoform. Primary rat neuronal cell cultures express predominantly the brain specific LCB2 isoform, whereas primary rat cultures of glia express only the LCB3 isoform, suggesting that expression of the brain-specific LCB2 form is limited to neurons. Further evidence for neuronal localization of the LCB2 form is provided using a teratocarcinoma cell line, P19, which can be induced by retinoic acid to express a neuronal phenotype, concomitant with the induction of the LCB2 form. In order to determine the sequences involved in alternative splice site selection, we constructed a minigene containing the alternative spliced exon EN and its flanking intron and exon sequences. This minigene reflects the splicing pattern of the endogenous gene upon transfection in HeLa cell and primary neuronal cell cultures, indicating that this region of the LCB gene contains all the necessary information for neuron-specific splicing. Images PMID:1408826

  18. Myosin light chain kinase-dependent microvascular hyperpermeability in thermal injury.

    PubMed

    Huang, Qiaobing; Xu, Wenjuan; Ustinova, Elena; Wu, Mack; Childs, Ed; Hunter, Felicia; Yuan, Sarah

    2003-10-01

    Although the critical role of systemic inflammatory edema in the development of multiple organ failure in patients with massive burns has been fully recognized, the precise mechanisms responsible for the accumulation of blood fluid and proteins in tissues remote from the burn wound are poorly understood. The aim of this study was to test the hypothesis that circulating factors released during thermal injury cause microvascular leakage by triggering endothelial cell contraction and barrier dysfunction. A third-degree scald burn was induced in rats on the dorsal skin covering 25% total body surface area. The microcirculation and transvascular flux of albumin were observed in the rat mesentery using intravital fluorescence microscopy. The direct effect of circulating factors on microvascular barrier function was assessed by measuring the apparent permeability coefficient of albumin in isolated rat mesenteric venules during perfusion of plasma freshly withdrawn from burned rats. The in vivo study showed that the transvenular flux of albumin was significantly increased over a 6-h period with a maximal response seen at 3 h postburn. Importantly, perfusion of noninjured venules with burn plasma induced a time-dependent increase in albumin permeability. Pharmacological inhibition of protein kinase C, Src tyrosine kinases, or mast cell activation did not significantly affect the hyperpermeability response; however, blockage of myosin light chain phosphorylation with the myosin light chain kinase inhibitor ML-7 greatly attenuated the burn-induced increase in venular permeability in a dose-related pattern. The results support a role for endogenous circulating factors in microvascular leakage during burns. Myosin light chain phosphorylation-dependent endothelial contractile response may serve as an end-point effector leading to microvascular barrier dysfunction. PMID:14501951

  19. High sensitivity and specificity of elevated cerebrospinal fluid kappa free light chains in suspected multiple sclerosis.

    PubMed

    Hassan-Smith, G; Durant, L; Tsentemeidou, A; Assi, L K; Faint, J M; Kalra, S; Douglas, M R; Curnow, S J

    2014-11-15

    Cerebrospinal fluid (CSF) analysis is routinely used in the diagnostic work-up of multiple sclerosis (MS), by detecting CSF-specific oligoclonal bands (OCB). More recently, several studies have reported CSF free light chains (FLC) as an alternative. We show that absolute CSF κFLC concentrations were highly sensitive - more than OCB testing - and specific for clinically isolated syndrome, relapsing remitting and primary progressive MS. Measurement of κFLC alone was sufficient. Our results suggest that CSF κFLC levels measured by nephelometry, if validated in a larger series, are a preferred test to OCB analysis in the diagnostic work-up of patients suspected of having MS.

  20. Characterization of DLC-A and DLC-B, two families of cytoplasmic dynein light chain subunits.

    PubMed Central

    Gill, S R; Cleveland, D W; Schroer, T A

    1994-01-01

    Cytoplasmic dynein is a minus-end-directed, microtubule-dependent motor composed of two heavy chains (approximately 530 kDa), three intermediate chains (approximately 74 kDa), and a family of approximately 52-61 kDa light chains. Although the approximately 530 kDa subunit contains the motor and microtubule binding domains of the complex, the functions of the smaller subunits are not known. Using two-dimensional gel electrophoresis and proteolytic mapping, we show here that the light chains are composed of two major families, a higher M(r) family (58, 59, 61 kDa; dynein light chain group A [DLC-A]) and lower M(r) family (52, 53, 55, 56 kDa; dynein light chain group B [DLC-B]). Dissociation of the cytoplasmic dynein complex with potassium iodide reveals that all light chain polypeptides are tightly associated with the approximately 530 kDa heavy chain, whereas the approximately 74 kDa intermediate chain polypeptides are more readily extracted. Treatment with alkaline phosphatase alters the mobility of four of the light chain polypeptides, indicating that these subunits are phosphorylated. Sequencing of a cDNA clone encoding one member of the DLC-A family reveals a predicted globular structure that is not homologous to any known protein but does contain numerous potential phosphorylation sites and a consensus nucleotide-binding motif. Images PMID:7949421

  1. Anti-cardiolipin/beta-2 glycoprotein activities co-exist on human anti-DNA antibody light chains.

    PubMed

    Kumar, Sanjeev; Nagl, Sylvia; Kalsi, J K; Ravirajan, C T; Athwal, Dee; Latchman, David S; Pearl, Laurence H; Isenberg, David A

    2003-12-01

    We have recently shown that the human anti-DNA antibodies B3 and 33H11 also bind cardiolipin and that the anti-autoantigen activity resides predominantly on their lambda light chains. We now show that the two auto-antibodies possess strong reactivity to the plasma-protein 2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 with insignificant reactivity to cardiolipin or to beta2-GPI, we now demonstrate that hybrid Fabs with constituent light chain, but not the heavy chain, of B3 or 33H11, exhibit anti-cardiolipin activity. Furthermore, the constructs possessing the auto-antibody-derived light chain also exhibited significant reactivity to beta2-GPI. The results suggest that anti-DNA, anti-cardiolipin and anti-beta2-GPI activities co-exist on the light chains of the antibodies studied and, importantly, these activities could be transferred to antibody constructs by their light chains alone. Computer-generated models of the three-dimensional structures of the auto-antibodies and their hybrids, suggest predominant interaction of their light chains with domain IV of beta2-GPI.

  2. Molecular analysis of the immunoglobulin genes in goose.

    PubMed

    Huang, Tian; Wu, Kun; Yuan, Xiaoli; Shao, Shuai; Wang, WenYuan; Wei, Si; Cao, Gengsheng

    2016-07-01

    Immunoglobulins play an important role in adaptive immune system as defense molecules against pathogens. However, our knowledge on avian immunoglobulin genes has been limited to a few species. In this study, we analyzed goose (Anser cygnoides orientalis) immunoglobulin genes. Three IgH classes including IgM, IgA, IgY and λ light chain were identified. The IgM and IgA heavy chain constant regions are characteristically similar to their counterparts described in other vertebrates. In addition to the classic Ig isotypes, we also detected a transcript that encoded a truncated form of IgY (IgY(ΔFc)) in goose. Similar to duck, the IgY(ΔFc) in goose was generated by using different transcriptional termination signal of the same υ gene. Limited variability and only one leader peptide were observed in VH and VL domains, which suggested that gene conversion was the primary mechanism involved in goose antibody diversity. Our study provides more insights into the immunoglobulin genes in goose that had not been fully explored before.

  3. Effect of Lysine Modification on the Stability and Cellular Binding of Human Amyloidogenic Light Chains

    SciTech Connect

    O'Neill, Hugh Michael; Davern, Sandra M.; Murphy, Charles L.; Wall, Jonathan; Deborah, Weiss T.; Solomon, Alan

    2011-01-01

    AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescent microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichorism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.

  4. Four structural risk factors identify most fibril-forming kappa light chains.

    SciTech Connect

    Stevens, F. J.; Biosciences Division

    2000-09-01

    Antibody light chains (LCs) comprise the most structurally diverse family of proteins involved in amyloidosis. Many antibody LCs incorporate structural features that impair their stability and solubility, leading to their assembly into fibrils and to their subsequent pathological deposition when produced in excess during multiple myeloma and primary amyloidosis. The particular amino acid variations in antibody LCs that account for fibril formation and amyloidogenesis have not been identified. This study focuses on amyloidogenesis within the Kl family of human LCs. Reanalysis of the current database of primary structures of proteins from more than 100 patients who produced Kl LCS, 37 of which were amyloidogenic, reveals apparent structural features that may contribute to amyloidosis. These features include loss of conserved residues or the gain of particular residues through mutation at sites involving a repertoire of approximately 20% of the amino acid positions in the light chain variable domain (V{sub L}). Moreover, 80% of all K1 amyloidogenic V{sub L}s are identifiable by the presence of at least one of three single-site substitutions or the acquisition of an N-linked glycosylation site through mutations. These findings suggest that it is feasible to predict fibril propensity by analysis of primary structure.

  5. CaMKII in addition to MLCK contributes to phosphorylation of regulatory light chain in cardiomyocytes.

    PubMed

    Eikemo, Hilde; Moltzau, Lise Román; Hussain, Rizwan I; Nguyen, Cam H T; Qvigstad, Eirik; Levy, Finn Olav; Skomedal, Tor; Osnes, Jan-Bjørn

    2016-02-26

    The aim was to identify kinase activities involved in the phosphorylation of regulatory light chain (RLC) in situ in cardiomyocytes. In electrically stimulated rat cardiomyocytes, phosphatase inhibition by calyculin A unmasked kinase activities evoking an increase of phosphorylated RLC (P-RLC) from about 16% to about 80% after 80 min. The phosphorylation rate in cardiomyocytes was reduced by about 40% by the myosin light chain kinase (MLCK) inhibitor, ML-7. In rat ventricular muscle strips, calyculin A induced a positive inotropic effect that correlated with P-RLC levels. The inotropic effect and P-RLC elevation were abolished by ML-7 treatment. The kinase activities phosphorylating RLC in cardiomyocytes were reduced by about 60% by the non-selective kinase inhibitor staurosporine and by about 50% by the calmodulin antagonist W7. W7 eliminated the inhibitory effect of ML-7, suggesting that the cardiac MLCK is Ca(2+)/calmodulin (CaM)-dependent. The CaM-dependent kinase II (CaMKII) inhibitor KN-93 attenuated the calyculin A-induced RLC phosphorylation by about 40%, indicating a contribution from CaMKII. The residual phosphorylation in the presence of W7 indicated that also CaM-independent kinase activities might contribute. RLC phosphorylation was insensitive to protein kinase C inhibition. In conclusion, in addition to MLCK, CaMKII phosphorylates RLC in cardiomyocytes. Involvement of other kinases cannot be excluded.

  6. The Roadblock light chains are ubiquitous components of cytoplasmic dynein that form homo- and heterodimers.

    PubMed

    Nikulina, Karina; Patel-King, Ramila S; Takebe, Sachiko; Pfister, K Kevin; King, Stephen M

    2004-04-01

    The Roadblock/LC7 class of light chains associate with the intermediate chains at the base of the soluble dynein particle. In mammals, there are two Roadblock isoforms (Robl1 and Robl2), one of which (Robl2) is differentially expressed in a tissue-dependent manner and is especially prominent in testis. Here we define the alpha helical content of Robl and demonstrate using both the yeast two-hybrid system and in vitro biochemistry that Robl1 and Robl2 are capable of forming homo- and heterodimers. This is the first report of heterodimer formation by any cytoplasmic dynein component, and it further enlarges the number of potential cytoplasmic dynein isoforms available for binding specific cellular cargoes. In addition, we have generated an antibody that specifically recognizes Robl light chains and shows a 5-10 fold preference for Robl2 over Robl1. Using this antibody, we show that Robl is a ubiquitous cytoplasmic dynein component, being found in samples purified from brain, liver, kidney, and testis. Immunofluorescence analysis reveals that Robl is present in punctate organelles in rat neuroblastoma cells. In testis, Robl is found in Leydig cells, spermatocytes, and sperm flagella.

  7. A Case of Abdominal Aortic Retroperitoneal and Mesenteric Amyloid Light Chain Amyloidoma

    PubMed Central

    Yokota, Kazuhiro; Kishida, Dai; Kayano, Hidekazu; Yazaki, Masahide; Shimada, Yuki; Akiyama, Yuji

    2016-01-01

    We report the case of a Japanese woman with amyloid light chain (AL) amyloidoma in the abdominal aortic retroperitoneum and mesentery. Irregular soft tissue mass lesions with calcification in the abdominal aortic retroperitoneum and mesentery were initially detected by computed tomography at another hospital. The lesions gradually compressed the duodenum, causing symptoms of bowel obstruction. The patient was clinically diagnosed with retroperitoneal fibrosis, and prednisolone was administered at a dose of 40 mg/day. However, the lesions did not change in size and her symptoms continued. She was transferred to our hospital and underwent mesenteric biopsy for histopathology using abdominal laparotomy. The histopathological and immunohistological findings of the mesenteric specimen demonstrated lambda light chain deposition. Accordingly, the patient was finally diagnosed with AL amyloidoma with no evidence of systemic amyloidosis. After laparotomy, her general condition worsened because of complications of pneumonia and deep vein thrombosis. She died suddenly from acute myocardial infarction. We have concluded that abdominal aortic retroperitoneal and mesenteric AL amyloidoma may have very poor prognoses in accordance with previous reports. In addition, the size and location of AL amyloidoma directly influence the prognosis. We suggest that early histopathology is important for improving prognosis. PMID:27752386

  8. Endothelial cell substrate stiffness influences neutrophil transmigration via myosin light chain kinase-dependent cell contraction.

    PubMed

    Stroka, Kimberly M; Aranda-Espinoza, Helim

    2011-08-11

    A vast amount of work has been dedicated to the effects of shear flow and cytokines on leukocyte transmigration. However, no studies have explored the effects of substrate stiffness on transmigration. Here, we investigated important aspects of endothelial cell contraction-mediated neutrophil transmigration using an in vitro model of the vascular endothelium. We modeled blood vessels of varying mechanical properties using fibronectin-coated polyacrylamide gels of varying physiologic stiffness, plated with human umbilical vein endothelial cell (HUVEC) monolayers, which were activated with tumor necrosis factor-α. Interestingly, neutrophil transmigration increased with increasing substrate stiffness below the endothelium. HUVEC intercellular adhesion molecule-1 expression, stiffness, cytoskeletal arrangement, morphology, and cell-substrate adhesion could not account for the dependence of transmigration on HUVEC substrate stiffness. We also explored the role of cell contraction and observed that large holes formed in endothelium on stiff substrates several minutes after neutrophil transmigration reached a maximum. Further, suppression of contraction through inhibition of myosin light chain kinase normalized the effects of substrate stiffness by reducing transmigration and eliminating hole formation in HUVECs on stiff substrates. These results provide strong evidence that neutrophil transmigration is regulated by myosin light chain kinase-mediated endothelial cell contraction and that this event depends on subendothelial cell matrix stiffness. PMID:21652678

  9. Clathrin light chains' role in selective endocytosis influences antibody isotype switching.

    PubMed

    Wu, Shuang; Majeed, Sophia R; Evans, Timothy M; Camus, Marine D; Wong, Nicole M L; Schollmeier, Yvette; Park, Minjong; Muppidi, Jagan R; Reboldi, Andrea; Parham, Peter; Cyster, Jason G; Brodsky, Frances M

    2016-08-30

    Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor β receptor 2 (TGFβR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the β2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules. PMID:27540116

  10. Structural and Thermodynamic Characterization of a Cytoplasmic Dynein Light Chain-Intermediate Chain Complex

    SciTech Connect

    Williams,J.; Roulhac, P.; Roy, A.; Vallee, R.; Fitzgerald, M.; Hendrickson, W.

    2007-01-01

    Cytoplasmic dynein is a microtubule-based motor protein complex that plays important roles in a wide range of fundamental cellular processes, including vesicular transport, mitosis, and cell migration. A single major form of cytoplasmic dynein associates with membranous organelles, mitotic kinetochores, the mitotic and migratory cell cortex, centrosomes, and mRNA complexes. The ability of cytoplasmic dynein to recognize such diverse forms of cargo is thought to be associated with its several accessory subunits, which reside at the base of the molecule. The dynein light chains (LCs) LC8 and TcTex1 form a subcomplex with dynein intermediate chains, and they also interact with numerous protein and ribonucleoprotein partners. This observation has led to the hypothesis that these subunits serve to tether cargo to the dynein motor. Here, we present the structure and a thermodynamic analysis of a complex of LC8 and TcTex1 associated with their intermediate chain scaffold. The intermediate chains effectively block the major putative cargo binding sites within the light chains. These data suggest that, in the dynein complex, the LCs do not bind cargo, in apparent disagreement with a role for LCs in dynein cargo binding interactions.

  11. Cloning and characterization of a dynein light chain gene from Puccinia striiformis f. sp. tritici.

    PubMed

    Liu, Jie; Zhang, Qiong; Chang, Qing; Wang, Qiuling; Han, Lina; Liu, Jia; Li, Man; Zhuang, Hua; Kang, Zhensheng

    2014-07-01

    Stripe rust is one of the most serious wheat diseases worldwide. The fungus Puccinia striiformis f. sp. tritici (Pst), the causal agent of this disease, is an obligate biotrophic basidiomycete fungus. Numerous studies have shown that dyneins play important roles during fungal growth and propagation. However, knowledge is limited regarding the function of dyneins in Pst. In this study, we cloned the dynein light chain gene PsDLC1 from Pst and characterized its expression. The function of PsDLC1 was determined by heterologous mutant complementation. Expression of PsDLC1 in Aspergillus nidulans partially complemented the defects of the ΔnudG mutant, indicating that PsDLC1 belongs to the dynein light chain LC8 family. In addition, PsDLC1 was identified in Pst using virus-induced gene silencing (VIGS). Knockdown of PsDLC1 produces no significant effect on Pst growth and development, indicating that PsDLC1 is unnecessary for Pst infection of wheat.

  12. Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase.

    PubMed

    Feghhi, Shirin; Tooley, Wes W; Sniadecki, Nathan J

    2016-10-01

    Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis. PMID:27548633

  13. High-level production of a functional immunoglobulin heterodimer in a baculovirus expression system.

    PubMed Central

    Hasemann, C A; Capra, J D

    1990-01-01

    A murine immunoglobulin heterodimer has been expressed in a baculovirus expression system. This was achieved by using both double infection of insect cells with separate heavy- and light-chain-expressing viruses and infection with a double-recombinant virus containing both the immunoglobulin heavy- and light-chain cDNAs. In both cases, the polypeptide chains were correctly processed, glycosylated, and assembled into normal H2L2 (H = heavy, L = light) immunoglobulin monomers. These molecules bound antigen and expressed both polyclonal idiotype and monoclonal idiotopes. Furthermore, the transfer vectors described have been modified to contain the F1 origin of replication for the production of single-stranded DNA, which facilitates site-specific mutations of either the polyhedrin promoter or the inserted foreign gene. Use of this system should significantly advance the analysis of the structural bases for both idiotype expression and antigen binding by immunoglobulin. More importantly, it provides a generic method for the high-level expression of antibodies of diverse interest. Images PMID:2111022

  14. Omega-3 fatty acids (image)

    MedlinePlus

    Omega-3 fatty acids are a form of polyunsaturated fat that the body derives from food. Omega-3s (and omega-6s) are known as essential fatty acids (EFAs) because they are important for good health. ...

  15. Structure–Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants

    PubMed Central

    Shen, Kui; Ramirez, Benjamin; Mapes, Brandon; Shen, Grace R.; Gokhale, Vijay; Brown, Mary E.; Santarsiero, Bernard; Ishii, Yoshitaka; Dudek, Steven M.; Wang, Ting; Garcia, Joe G. N.

    2015-01-01

    The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities. PMID:26111161

  16. Follow-up of IgD-κ multiple myeloma by monitoring free light chains and total heavy chain IgD: A case report

    PubMed Central

    De Santis, Elena; Masi, Serena; Cordone, Iole; Pisani, Francesco; Zuppi, Cecilia; Mattei, Fabrizio; Conti, Laura; Cigliana, Giovanni

    2016-01-01

    Immunoglobulin (Ig)D-κ multiple myeloma (MM) is a rare neoplastic disease characterized by an aggressive and rapidly progressing course, which constitutes only a very small proportion of all MM cases. In the present report, the clinical case of a 51-year-old Caucasian woman diagnosed with IgD-κ MM is described. The patient underwent different chemotherapeutic treatments subsequently to a single autologous stem cell transplantation. Despite the inherent difficulty of monitoring IgD levels and performing serum immunofixation electrophoresis, the clinical outcome of the patient was almost uniquely monitored by measuring the levels of κ and λ free light chains (FLCs) and total heavy chain IgD. The data suggest the non-invasive potential and usefulness of FLCs evaluation for early detection of stringent complete remission, follow-up and early detection of disease relapse. In addition, this diagnostic procedure has successfully been employed for the therapeutic monitoring of the present patient, and may represent a very helpful, non-invasive tool for the follow-up of IgD myeloma patients without the requirement of serial bone marrow aspirate. PMID:27588135

  17. Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants.

    PubMed

    Shen, Kui; Ramirez, Benjamin; Mapes, Brandon; Shen, Grace R; Gokhale, Vijay; Brown, Mary E; Santarsiero, Bernard; Ishii, Yoshitaka; Dudek, Steven M; Wang, Ting; Garcia, Joe G N

    2015-01-01

    The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

  18. Sequencing of the human IG light chain loci from a hydatidiform mole BAC library reveals locus-specific signatures of genetic diversity

    PubMed Central

    Graves, Tina A.; Warren, Rene L.; Malig, Maika; Schein, Jacqueline; Wilson, Richard K.; Holt, Robert A.; Eichler, Evan E.; Breden, Felix

    2014-01-01

    Germline variation at immunoglobulin (IG) loci is critical for pathogen-mediated immunity, but establishing complete haplotype sequences in these regions has been problematic because of complex sequence architecture and diploid source DNA. We sequenced BAC clones from the effectively haploid human hydatidiform mole cell line, CHM1htert, across the light chain IG loci, kappa (IGK) and lambda (IGL), creating single haplotype representations of these regions. The IGL haplotype generated here is 1.25 Mb of contiguous sequence, including four novel V alleles and one novel C allele and an 11.9 kb insertion. The CH17 IGK haplotype consists of two 644 kb proximal and 466 kb distal contigs separated by a large gap of unknown size; these assemblies added 49 kb of unique sequence extending into this gap. Our analysis also resulted in the characterization of seven novel IGKV alleles and a 16.7 kb region exhibiting signatures of interlocus sequence exchange between distal and proximal IGKV gene clusters. Genetic diversity in IGK/IGL was compared to that of the IG heavy chain (IGH) locus within the same haploid genome, revealing 3-fold (IGK) and 6-fold (IGL) higher diversity in the IGH locus, potentially associated with increased levels of segmental duplication and the telomeric location of IGH. PMID:25338678

  19. Omega-3 Fatty Acids

    MedlinePlus

    Omega-3 fatty acids are used together with lifestyle changes (diet, weight-loss, exercise) to reduce the amount of triglycerides (a fat-like ... people with very high triglycerides. Omega-3 fatty acids are in a class of medications called antilipemic ...

  20. Retina and Omega-3

    PubMed Central

    Querques, Giuseppe; Forte, Raimondo; Souied, Eric H.

    2011-01-01

    Over the last decade, several epidemiological studies based on food frequency questionnaires suggest that omega-3 polyunsaturated fatty acids could have a protective role in reducing the onset and progression of retinal diseases. The retina has a high concentration of omega-3, particularly DHA, which optimizes fluidity of photoreceptor membranes, retinal integrity, and visual function. Furthermore, many studies demonstrated that DHA has a protective, for example antiapoptotic, role in the retina. From a nutritional point of view, it is known that western populations, particularly aged individuals, have a higher than optimal omega-6/omega-3 ratio and should enrich their diet with more fish consumption or have DHA supplementation. This paper underscores the potential beneficial effect of omega-3 fatty acids on retinal diseases. PMID:22175009

  1. Stochastic rearrangement of immunoglobulin variable-region genes in chicken B-cell development.

    PubMed Central

    Benatar, T; Tkalec, L; Ratcliffe, M J

    1992-01-01

    The molecular mechanism by which immunoglobulin (Ig) gene rearrangement occurs is highly conserved between mammalian and avian species. However, in avian species, an equivalent to the mammalian pre-B cell, which has undergone Ig heavy-chain gene rearrangement and expresses mu heavy chains in the absence of Ig light-chain rearrangement, has not been convincingly demonstrated. It is consequently unclear whether an ordered progression of gene rearrangement events leading to functional Ig expression occurs in avian species. To examine the sequence of Ig gene rearrangement events in chicken B-cell development, we transformed day 12 embryo bursal cells with the REV-T(CSV) retrovirus. More than 100 clones were analyzed by Southern blotting and polymerase chain reaction for the presence of Ig gene rearrangements. The majority of these clones contained only germline Ig sequences. Several clones contained complete heavy- and light-chain rearrangements and 13 clones contained only heavy-chain rearrangements analogous to stages of mammalian B-cell development. However, 5 clones contained rearrangements of light-chain genes in the absence of complete heavy-chain rearrangement. Consequently, we conclude that rearrangement of chicken Ig light-chain genes does not require heavy-chain variable-region rearrangement. This observation suggests that chicken Ig gene rearrangement events required for Ig expression occur stochastically rather than sequentially. Images PMID:1502173

  2. Metachronous/concomitant B-cell neoplasms with discordant light-chain or heavy-chain isotype restrictions: evidence of distinct B-cell neoplasms rather than clonal evolutions.

    PubMed

    Wei, Qiang; Sebastian, Siby; Papavassiliou, Paulie; Rehder, Catherine; Wang, Endi

    2014-10-01

    Metachronous/concomitant B-cell neoplasms with distinct morphology are usually considered clonally related. We retrospectively analyzed 4 cases of metachronous/concomitant B-cell neoplasms with discordant light-chain/heavy-chain restrictions. The primary diagnoses included chronic lymphocytic leukemia (CLL; n = 2), lymphoplasmacytic lymphoma (n = 1), and pediatric follicular lymphoma (FL; n = 1). The respective secondary diagnoses included diffuse large B-cell lymphoma (DLBCL; n = 2), plasmablastic myeloma, and pediatric FL. The secondary B-cell neoplasm occurred after the primary diagnosis in 3 cases, with the median interval of 120 months (range, 21-216), whereas the remaining 1 case had the 2 neoplasms (CLL/DLBCL) diagnosed concurrently. Histology suggested aggressive transformation in 3 cases and recurrence in 1 case (FL). Nonetheless, 3 cases showed discordant light-chain restrictions between the 2 B-cell neoplasms, whereas in the remaining case (lymphoplasmacytic lymphoma/plasmablastic myeloma), the 2 neoplasms shared κ light-chain restriction but expressed different heavy-chain isotypes (IgM versus IgA). The 2 CLL/DLBCL cases had polymerase chain reaction-based IGH/K gene rearrangement study and amplicon sequence analysis performed, which demonstrated distinct clonal amplicons between the 2 B-cell neoplasms in each case. Concomitant/metachronous B-cell neoplasms may be clonally unrelated, which can be confirmed by immunoglobulin isotype analysis and/or genotypic studies. We advocate analysis of clonal identities in large cell transformation or recurrent disease compared with primary indolent B-cell neoplasm because of a potential difference in prognosis between clonally related and unrelated secondary B-cell neoplasms.

  3. Purification, Characterization, and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkii.

    PubMed

    Zhang, Yong-Xia; Chen, Heng-Li; Maleki, Soheila J; Cao, Min-Jie; Zhang, Ling-Jing; Su, Wen-Jin; Liu, Guang-Ming

    2015-07-15

    Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with a molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali, and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.

  4. AL (Light-Chain) Cardiac Amyloidosis: A Review of Diagnosis and Therapy.

    PubMed

    Falk, Rodney H; Alexander, Kevin M; Liao, Ronglih; Dorbala, Sharmila

    2016-09-20

    The amyloidoses are a group of protein-folding disorders in which ≥1 organ is infiltrated by proteinaceous deposits known as amyloid. The deposits are derived from 1 of several amyloidogenic precursor proteins, and the prognosis of the disease is determined both by the organ(s) involved and the type of amyloid. Amyloid involvement of the heart (cardiac amyloidosis) carries the worst prognosis of any involved organ, and light-chain (AL) amyloidosis is the most serious form of the disease. The last decade has seen considerable progress in understanding the amyloidoses. In this review, current and novel approaches to the diagnosis and treatment of cardiac amyloidosis are discussed, with particular reference to AL amyloidosis in the heart. PMID:27634125

  5. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  6. Recurrent Syncope and Cardiac Arrest in a Patient with Systemic Light Chain Amyloidosis Treated with Bortezomib.

    PubMed

    Jaipaul, Navin; Pi, Alexander; Zhang, Zhiwei

    2016-05-10

    About 10-15% of patients with multiple myeloma develop light chain (AL) amyloidosis. AL amyloidosis is a systemic disease that may involve multiple organs, often including the heart. It may present clinically with bradyarrhythmia and syncope. The proteasome inhibitor bortezomib has been used with clinical efficacy in treating patients with AL amyloidosis but also implicated as a possible cause of cardiomyocyte injury. We report a case of a 48-year-old man with AL amyloidosis and increased frequency of syncope and cardiac arrest after starting bortezomib. The biologic and clinical plausibility of a heightened risk for cardiac arrest in patients with cardiac AL amyloidosis and history of syncope being treated with bortezomib is a possibility that is not well documented in the medical literature and warrants further investigation. PMID:27499835

  7. Recurrent Syncope and Cardiac Arrest in a Patient with Systemic Light Chain Amyloidosis Treated with Bortezomib

    PubMed Central

    Jaipaul, Navin; Pi, Alexander; Zhang, Zhiwei

    2016-01-01

    About 10-15% of patients with multiple myeloma develop light chain (AL) amyloidosis. AL amyloidosis is a systemic disease that may involve multiple organs, often including the heart. It may present clinically with bradyarrhythmia and syncope. The proteasome inhibitor bortezomib has been used with clinical efficacy in treating patients with AL amyloidosis but also implicated as a possible cause of cardiomyocyte injury. We report a case of a 48-year-old man with AL amyloidosis and increased frequency of syncope and cardiac arrest after starting bortezomib. The biologic and clinical plausibility of a heightened risk for cardiac arrest in patients with cardiac AL amyloidosis and history of syncope being treated with bortezomib is a possibility that is not well documented in the medical literature and warrants further investigation. PMID:27499835

  8. Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle

    PubMed Central

    1988-01-01

    The time course of [Ca2+]i, tension, and myosin light chain phosphorylation were determined during prolonged depolarization with high K+ in intact tonic (rabbit pulmonary artery) and phasic (longitudinal layer of guinea pig ileum) smooth muscles. [Ca2+]i was monitored with the 340 nm/380 nm signal ratio of the fluorescent indicator fura-2. The fluorescence ratio had a similar time course in both muscle types during depolarization with 109 mM [K+]o; after a transient peak, there was a decline to 70% of its peak value in tonic smooth muscle, and to 60% in phasic smooth muscle. Tension, however, continued to increase in the pulmonary artery, while in the ileum it declined in parallel with the [Ca2+]i. On changing [K+]o from 109 to 20 mM, tension and [Ca2+]i either remained unchanged or declined in parallel in the pulmonary artery. Phosphorylation of the 20-kD myosin light chain, measured during stimulation of muscle strips with 109 mM [K+]o in another set of experiments, increased from 3% to a peak of 50% in the intact pulmonary artery, and then declined to a steady state value of 23%. In the intact ileum, a very rapid, early transient phosphorylation (up to 50%) at 2-3 s was seen. This transient declined by 30 s to a value that was close to the resting level (7%), while tension remained at 55% of its peak force. A quick release during maintained stimulation induced no detectable change in the [Ca2+]i in either type of smooth muscle. We discuss the possibility that the slowly rising tonic tension in pulmonary artery could be due to cooperativity between phosphorylated and nonphosphorylated crossbridges. PMID:3216188

  9. Crystal Structures of the Tetratricopeptide Repeat Domains of Kinesin Light Chains: Insight into Cargo Recognition Mechanisms

    SciTech Connect

    Zhu, Haizhong; Lee, Han Youl; Tong, Yufeng; Hong, Bum-Soo; Kim, Kyung-Phil; Shen, Yang; Lim, Kyung Jik; Mackenzie, Farrell; Tempel, Wolfram; Park, Hee-Won

    2012-10-23

    Kinesin-1 transports various cargos along the axon by interacting with the cargos through its light chain subunit. Kinesin light chains (KLC) utilize its tetratricopeptide repeat (TPR) domain to interact with over 10 different cargos. Despite a high sequence identity between their TPR domains (87%), KLC1 and KLC2 isoforms exhibit differential binding properties towards some cargos. We determined the structures of human KLC1 and KLC2 tetratricopeptide repeat (TPR) domains using X-ray crystallography and investigated the different mechanisms by which KLCs interact with their cargos. Using isothermal titration calorimetry, we attributed the specific interaction between KLC1 and JNK-interacting protein 1 (JIP1) cargo to residue N343 in the fourth TRP repeat. Structurally, the N343 residue is adjacent to other asparagines and lysines, creating a positively charged polar patch within the groove of the TPR domain. Whereas, KLC2 with the corresponding residue S328 did not interact with JIP1. Based on these finding, we propose that N343 of KLC1 can form 'a carboxylate clamp' with its neighboring asparagine to interact with JIP1, similar to that of HSP70/HSP90 organizing protein-1's (HOP1) interaction with heat shock proteins. For the binding of cargos shared by KLC1 and KLC2, we propose a different site located within the groove but not involving N343. We further propose a third binding site on KLC1 which involves a stretch of polar residues along the inter-TPR loops that may form a network of hydrogen bonds to JIP3 and JIP4. Together, these results provide structural insights into possible mechanisms of interaction between KLC TPR domains and various cargo proteins.

  10. PKC-mediated cerebral vasoconstriction: Role of myosin light chain phosphorylation versus actin cytoskeleton reorganization.

    PubMed

    El-Yazbi, Ahmed F; Abd-Elrahman, Khaled S; Moreno-Dominguez, Alejandro

    2015-06-15

    Defective protein kinase C (PKC) signaling has been suggested to contribute to abnormal vascular contraction in disease conditions including hypertension and diabetes. Our previous work on agonist and pressure-induced cerebral vasoconstriction implicated PKC as a major contributor to force production in a myosin light chain (LC20) phosphorylation-independent manner. Here, we used phorbol dibutyrate to selectively induce a PKC-dependent constriction in rat middle cerebral arteries and delineate the relative contribution of different contractile mechanisms involved. Specifically, we employed an ultra-sensitive 3-step western blotting approach to detect changes in the content of phosphoproteins that regulate myosin light chain phosphatase (MLCP) activity, thin filament activation, and actin cytoskeleton reorganization. Data indicate that PKC activation evoked a greater constriction at a similar level of LC20 phosphorylation achieved by 5-HT. PDBu-evoked constriction persisted in the presence of Gö6976, a selective inhibitor of Ca(2+)-dependent PKC, and in the absence of extracellular Ca(2+). Biochemical evidence indicates that either + or - extracellular Ca(2+), PDBu (i) inhibits MLCP activity via the phosphorylation of myosin targeting subunit of myosin phosphatase (MYPT1) and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17), (ii) increases the phosphorylation of paxillin and heat shock protein 27 (HSP27), and reduces G-actin content, and (iii) does not change the phospho-content of the thin filament proteins, calponin and caldesmon. PDBu-induced constriction was more sensitive to disruption of actin cytoskeleton compared to inhibition of cross-bridge cycling. In conclusion, this study provided evidence for the pivotal contribution of cytoskeletal actin polymerization in force generation following PKC activation in cerebral resistance arteries. PMID:25931148

  11. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon.

    PubMed

    Del Coso, Juan; Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts.

  12. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon

    PubMed Central

    Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts. PMID:27483374

  13. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon.

    PubMed

    Del Coso, Juan; Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts. PMID:27483374

  14. Light chain editing in kappa-deficient animals: a potential mechanism of B cell tolerance

    PubMed Central

    1994-01-01

    The genetic organization of the kappa and lambda light chain loci permits multiple, successive rearrangement attempts at each allele. Multiple rearrangements allow autoreactive B cells to escape clonal deletion by editing their surface receptors. Editing may also facilitate efficient B cell production by salvaging cells with nonproductive light chain (L chain) rearrangements. To study receptor editing of kappa L chains, we have characterized B cells from mice hemizygous for the targeted inactivation of kappa (JCkD/wt) which have an anti-DNA heavy chain transgene, 3H9. Hybridomas from JCkD/wt mice exhibited an increased frequency of rearrangements to downstream Jk segments (such as Jk5) compared with most surveys from normal mice, consistent with receptor editing by sequential kappa locus rearrangements in JCkD/wt. We observed an even higher frequency of rearrangements to Jk5 in 3H9 JCkD/wt animals compared with nontransgenic JCkD/wt, consistent with editing of autoreactive kappa in 3H9 JCkD/wt. We also recovered a large number of 3H9 JCkD/wt lines with Vk12/13-Jk5 rearrangements and could demonstrate by PCR and Southern analysis that up to three quarters of these lines underwent multiple kappa rearrangements. To investigate editing at the lambda locus, we used homozygous kappa-deficient animals (JCkD/JCkD and 3H9 JCkD/JCkD). The frequencies of V lambda 1 and V lambda 2 rearrangements among splenic hybridomas in 3H9 JCkD/JCkD were reduced by 75% whereas V lambda X was increased 5-10-fold, compared with nontransgenic JCkD/JCkD animals. This indicates that V lambda 1 and V lambda 2 are negatively regulated in 3H9 JCkD/JCkD, consistent with earlier studies that showed that the 3H9 heavy chain, in combination with lambda 1 binds DNA. As successive lambda rearrangements to V lambda X do not inactivate V lambda 1, the consequence of lambda editing in 3H9 JCkD/JCkD would be failed allelic exclusion at lambda. However, analysis of 18 3H9 JCkD/JCkD hybridomas with V lambda 1

  15. Clinical responses with T lymphocytes targeting malignancy-associated κ light chains

    PubMed Central

    Ramos, Carlos A.; Savoldo, Barbara; Torrano, Vicky; Ballard, Brandon; Zhang, Huimin; Dakhova, Olga; Liu, Enli; Carrum, George; Kamble, Rammurti T.; Gee, Adrian P.; Mei, Zhuyong; Wu, Meng-Fen; Liu, Hao; Grilley, Bambi; Rooney, Cliona M.; Brenner, Malcolm K.; Heslop, Helen E.; Dotti, Gianpietro

    2016-01-01

    BACKGROUND. Treatment of B cell malignancies with adoptive transfer of T cells with a CD19-specific chimeric antigen receptor (CAR) shows remarkable clinical efficacy. However, long-term persistence of T cells targeting CD19, a pan–B cell marker, also depletes normal B cells and causes severe hypogammaglobulinemia. Here, we developed a strategy to target B cell malignancies more selectively by taking advantage of B cell light Ig chain restriction. We generated a CAR that is specific for the κ light chain (κ.CAR) and therefore recognizes κ-restricted cells and spares the normal B cells expressing the nontargeted λ light chain, thus potentially minimizing humoral immunity impairment. METHODS. We conducted a phase 1 clinical trial and treated 16 patients with relapsed or refractory κ+ non-Hodgkin lymphoma/chronic lymphocytic leukemia (NHL/CLL) or multiple myeloma (MM) with autologous T cells genetically modified to express κ.CAR (κ.CARTs). Other treatments were discontinued in 11 of the 16 patients at least 4 weeks prior to T cell infusion. Six patients without lymphopenia received 12.5 mg/kg cyclophosphamide 4 days before κ.CART infusion (0.2 × 108 to 2 × 108 κ.CARTs/m2). No other lymphodepletion was used. RESULTS. κ.CART expansion peaked 1–2 weeks after infusion, and cells remained detectable for more than 6 weeks. Of 9 patients with relapsed NHL or CLL, 2 entered complete remission after 2 and 3 infusions of κ.CARTs, and 1 had a partial response. Of 7 patients with MM, 4 had stable disease lasting 2–17 months. No toxicities attributable to κ.CARTs were observed. CONCLUSION. κ.CART infusion is feasible and safe and can lead to complete clinical responses. TRIAL REGISTRATION. ClinicalTrials.gov NCT00881920. FUNDING. National Cancer Institute (NCI) grants 3P50CA126752 and 5P30CA125123 and Leukemia and Lymphoma Society (LLS) Specialized Centers of Research (SCOR) grant 7018. PMID:27270177

  16. Serum-free light-chain analysis in diagnosis and management of multiple myeloma and related conditions.

    PubMed

    Milani, Paolo; Palladini, Giovanni; Merlini, Giampaolo

    2016-01-01

    The introduction of the serum-free light-chain (S-FLC) assay has been a breakthrough in the diagnosis and management of plasma cell dyscrasias, particularly monoclonal light-chain diseases. The first method, proposed in 2001, quantifies serum-free light-chains using polyclonal antibodies. More recently, assays based on monoclonal antibodies have entered into clinical practice. S-FLC measurement plays a central role in the screening for multiple myeloma and related conditions, in association with electrophoretic techniques. Analysis of S-FLC is essential in assessing the risk of progression of precursor diseases to overt plasma cell dyscrasias. It is also useful for risk stratification in solitary plasmacytoma and AL amyloidosis. The S-FLC measurement is part of the new diagnostic criteria for multiple myeloma, and provides a marker to follow changes in clonal substructure over time. Finally, the evaluation of S-FLC is fundamental for assessing the response to treatment in monoclonal light chain diseases. PMID:27467897

  17. Immunoglobulin λ Gene Rearrangement Can Precede κ Gene Rearrangement

    DOE PAGES

    Berg, Jörg; Mcdowell, Mindy; Jäck, Hans-Martin; Wabl, Matthias

    1990-01-01

    Imore » mmunoglobulin genes are generated during differentiation of B lymphocytes by joining gene segments. A mouse pre-B cell contains a functional immunoglobulin heavy-chain gene, but no light-chain gene. Although there is only one heavy-chain locus, there are two lightchain loci: κ and λ .It has been reported that κ loci in the germ-line configuration are never (in man) or very rarely (in the mouse) present in cells with functionally rearranged λ -chain genes. Two explanations have been proposed to explain this: (a) the ordered rearrangement theory, which postulates that light-chain gene rearrangement in the pre-B cell is first attempted at the κ locus, and that only upon failure to produce a functional κ chain is there an attempt to rearrange the λ locus; and (b) the stochastic theory, which postulates that rearrangement at the λ locus proceeds at a rate that is intrinsically much slower than that at the κ locus. We show here that λ -chain genes are generated whether or not the κ locus has lost its germ-line arrangement, a result that is compatible only with the stochastic theory.« less

  18. Organization of immunoglobulin genes.

    PubMed

    Tonegawa, S; Brack, C; Hozumi, N; Pirrotta, V

    1978-01-01

    The nucleotide-sequence determination of a cloned, embryonic Vlambda gene directly demonstrated that V genes are separate from a corresponding C gene in embryonic cells. Analysis by restriction enzymes of total cellular DNA from various sources strongly suggested that the two separate immunoglobulin genes become continuous during differentiation of B lymphocytes. There seems to be a strict correlation between the joining event and activation of the joined genes. Cloning of more immunoglobulin genes from embryo and plasma cells will not only provide direct demonstration of such a gene-joining event but also help in the elucidation of a possible relationship of the event to gene activation mechanisms.

  19. Allelic exclusion of immunoglobulin genes: models and mechanisms.

    PubMed

    Vettermann, Christian; Schlissel, Mark S

    2010-09-01

    The allelic exclusion of immunoglobulin (Ig) genes is one of the most evolutionarily conserved features of the adaptive immune system and underlies the monospecificity of B cells. While much has been learned about how Ig allelic exclusion is established during B-cell development, the relevance of monospecificity to B-cell function remains enigmatic. Here, we review the theoretical models that have been proposed to explain the establishment of Ig allelic exclusion and focus on the molecular mechanisms utilized by developing B cells to ensure the monoallelic expression of Ig kappa and Ig lambda light chain genes. We also discuss the physiological consequences of Ig allelic exclusion and speculate on the importance of monospecificity of B cells for immune recognition.

  20. Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium.

    PubMed

    Pulcastro, Hannah C; Awinda, Peter O; Breithaupt, Jason J; Tanner, Bertrand C W

    2016-07-01

    Myosin force production is Ca(2+)-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca(2+)-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca(2+)-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 μm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 μm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles. PMID:26763941

  1. Sequence analysis of the myosin regulatory light chain gene of the vestimentiferan Riftia pachyptila.

    PubMed

    Ravaux, J; Hassanin, A; Deutsch, J; Gaill, F; Markmann-Mulisch, U

    2001-01-24

    We have isolated and characterized a cDNA (DNA complementary to RNA) clone (Rf69) from the vestimentiferan Riftia pachyptila. The cDNA insert consists of 1169 base pairs. The aminoacid sequence deduced from the longest reading frame is 193 residues in length, and clearly characterized it as a myosin regulatory light chain (RLC). The RLC primary structure is described in relation to its function in muscle contraction. The comparison with other RLCs suggested that Riftia myosin is probably regulated through its RLC either by phosphorylation like the vertebrate smooth muscle myosins, and/or by Ca2+-binding like the mollusk myosins. Riftia RLC possesses a N-terminal extension lacking in all other species besides the earthworm Lumbricus terrestris. Aminoacid sequence comparisons with a number of RLCs from vertebrates and invertebrates revealed a relatively high identity score (64%) between Riftia RLC and the homologous gene from Lumbricus. The relationships between the members of the myosin RLCs were examined by two phylogenetic methods, i.e. distance matrix and maximum parsimony. The resulting trees depict the grouping of the RLCs according to their role in myosin activity regulation. In all trees, Riftia RLC groups with RLCs that depend on Ca2+-binding for myosin activity regulation. PMID:11223252

  2. Kinesin's light chains inhibit the head- and microtubule-binding activity of its tail.

    PubMed

    Wong, Yao Liang; Rice, Sarah E

    2010-06-29

    Kinesin-1 is a microtubule-based motor comprising two heavy chains (KHCs) and two light chains (KLCs). Motor activity is precisely regulated to avoid futile ATP consumption and to ensure proper intracellular localization of kinesin-1 and its cargoes. The KHC tail inhibits ATPase activity by interacting with the enzymatic KHC heads, and the tail also binds microtubules. Here, we present a role for the KLCs in regulating both the head- and microtubule-binding activities of the kinesin-1 tail. We show that KLCs reduce the affinity of the head-tail interaction over tenfold and concomitantly repress the tail's regulatory activity. We also show that KLCs inhibit tail-microtubule binding by a separate mechanism. Inhibition of head-tail binding requires steric and electrostatic factors. Inhibition of tail-microtubule binding is largely electrostatic, pH dependent, and mediated partly by a highly negatively charged linker region between the KHC-interacting and cargo-binding domains of the KLCs. Our data support a model wherein KLCs promote activation of kinesin-1 for cargo transport by simultaneously suppressing tail-head and tail-microtubule interactions. KLC-mediated inhibition of tail-microtubule binding may also influence diffusional movement of kinesin-1 on microtubules, and kinesin-1's role in microtubule transport/sliding. PMID:20547877

  3. Effects of a Fluorescent Myosin Light Chain Phosphatase Inhibitor on Prostate Cancer Cells

    PubMed Central

    Grindrod, Scott; Suy, Simeng; Fallen, Shannon; Eto, Masumi; Toretsky, Jeffrey; Brown, Milton L.

    2011-01-01

    Myosin light chain phosphatase (MLCP) is an enzyme important to regulation of cell cycle and motility that is shown to be upregulated in aggressive prostate cancer cells and tissue. We developed a fluorescent small molecule inhibitor of MLCP using structure based design in recombinant protein phosphatase 1C. Several best fit compounds were synthesized and evaluated by their inhibition of MLCP/32P-MLC dephosphorylation, which resulted in the identification of novel MLCP inhibitors. Androgen dependent (AD) and castration resistant prostate cancer cell (CRPC) lines were treated with the lead inhibitor resulting in decreased growth rate, reduced DNA synthesis, and G2/M cell cycle arrest. Moreover, CRPC cell lines showed an increased sensitivity to drug treatment having GI50 values four times lower than the AD prostate cancer cell line. This was reinforced by reduced BrdU DNA incorporation into CRPC cells compared to AD cells. β-actin disruption was also seen at much lower drug concentrations in CR cells which caused a dose dependent reduction in cellular chemotaxis of PC-3 cells. Since there are currently few clinical therapeutics targeting CR prostate cancer, MLCP represents a new target for preclinical and clinical development of new potential therapeutics which inhibit this disease phenotype. PMID:22655237

  4. Functions of Myosin Light Chain-2 (MYL2) In Cardiac Muscle and Disease

    PubMed Central

    Sheikh, Farah; Lyon, Robert C.; Chen, Ju

    2015-01-01

    Myosin light chain-2 (MYL2, also called MLC-2) is an ∼19 kDa sarcomeric protein that belongs to the EF-hand calcium binding protein superfamily and exists as three major isoforms encoded by three distinct genes in mammalian striated muscle. Each of the three different MLC-2 genes (MLC-2f; fast twitch skeletal isoform, MLC-2v; cardiac ventricular and slow twitch skeletal isoform, MLC-2a; cardiac atrial isoform) has a distinct developmental expression pattern in mammals. Genetic loss-of-function studies in mice demonstrated an essential role for cardiac isoforms of MLC-2, MLC-2v and MLC-2a, in cardiac contractile function during early embryogenesis. In the adult heart, MLC-2v function is regulated by phosphorylation, which displays a specific expression pattern (high in epicardium and low in endocardium) across the heart. These data along with new data from computational models, genetic mouse models, and human studies have revealed a direct role for MLC-2v phosphorylation in cross-bridge cycling kinetics, calcium-dependent cardiac muscle contraction, cardiac torsion, cardiac function and various cardiac diseases. This review focuses on the regulatory functions of MLC-2 in the embryonic and adult heart, with an emphasis on phosphorylation-driven actions of MLC-2v in adult cardiac muscle, which provide new insights into mechanisms regulating myosin cycling kinetics and human cardiac diseases. PMID:26074085

  5. Regulatory and structural motifs of chicken gizzard myosin light chain kinase.

    PubMed Central

    Olson, N J; Pearson, R B; Needleman, D S; Hurwitz, M Y; Kemp, B E; Means, A R

    1990-01-01

    The amino acid sequence for chicken smooth muscle myosin light chain kinase (smMLCK) was deduced from a full-length cDNA. This has allowed definition of both the complete sequence of the inactive 64-kDa proteolytic fragment, which contains the pseudosubstrate autoregulatory sequence, and of the active 61-kDa Ca2+/calmodulin-independent fragment, which lacks the autoregulatory domain. Comparison of the two sequences shows that the autoregulatory domain extends from Asn-780 to Arg-808. The peptide Leu-774 to Ser-787 does not inhibit smMLCK, whereas peptides of similar or shorter length from the pseudosubstrate region (Ser-787 to Val-807) are potent inhibitors. These data define the autoregulatory region as being contained within and probably identical to the pseudosubstrate domain. The catalytic and regulatory regions are flanked by several copies of 100-amino acid segments containing one of two consensus motifs. These motifs are absent from mammalian skeletal muscle MLCK or from Dictyostelium discoideum MLCK but are present in the Caenorhabditis elegans unc-22 gene product and the titin molecule of skeletal muscle myofibrils. These results indicate that the amino acid sequence of smMLCK encodes multiple functional motifs in addition to the catalytic domain. PMID:2315320

  6. Prognostic value of serum heavy/light chain ratios in patients with POEMS syndrome.

    PubMed

    Wang, Chen; Su, Wei; Cai, Qian-Qian; Cai, Hao; Ji, Wei; Di, Qian; Duan, Ming-Hui; Cao, Xin-Xin; Zhou, Dao-Bin; Li, Jian

    2016-07-01

    POEMS syndrome is a rare plasma cell dyscrasia. Serum concentrations of the monoclonal protein in this disorder are typically low, and inapplicable to monitor disease activity in most cases, resulting in limited practical and prognostic values. Novel immunoassays measuring isotype-specific heavy/light chain (HLC) pairs showed its utility in disease monitoring and outcome prediction in several plasma cell dyscrasias. We report results of HLC measurements in 90 patients with POEMS syndrome. Sixty-six patients (73%; 95% confidence interval, 63-82%) had an abnormal HLC ratio at baseline. It could stratify the risk of disease relapse and was strongly associated with worse progression-free survival in a multivariate analysis (P = 0.021; hazard ratio [HR] 6.89, 95% CI 1.34-35.43). After therapy, HLC ratios improved, with 43 patients (48%) remaining abnormal. The post-therapeutic HLC ratio, if abnormal, also remained as an independent prognostic factor associated with worse progression-free survival (P = 0.019; HR 4.30, 95% CI 1.27-14.56). These results suggest the prognostic utility of HLC ratios in clinical management of POEMS patients.

  7. Mn2+ activates skinned smooth muscle cells in the absence of myosin light chain phosphorylation.

    PubMed

    Hoar, P E; Kerrick, W G

    1988-08-01

    Two effects of Mn2+ on skinned fibers from chicken gizzard smooth muscle were observed, dependent on the presence or absence of dithiothreitol (DTT) reducing agent. One involves protein oxidation (in the absence of DTT) with production of a "latch"-like state, and the other involves direct Mn2+ activation of contractile proteins. Cells activated by Mn2+ in the presence of ATP and the absence of Ca2+, Mg2+ and DTT did not relax when transferred to normal relaxing solutions. In contrast, when 5 mM DTT was included in the Mn2+ contracting solution to prevent protein oxidation by Mn2+, the cells still contracted when exposed to Mn2+, but relaxed rapidly when the Mn2+ was removed. In the presence of DTT both the Mn2+ activation and the relaxation following removal of Mn2+ were more rapid than normal Ca2+-activated contractions and relaxations. The skinned fibers activated by Mn2+ in the absence of DTT showed little active shortening unless DTT was added. This rigor-like state is probably due to oxidation of contractile proteins since the cells relaxed when exposed to a relaxing solution containing DTT (50 mM) and then contracted again in response to Ca2+ and relaxed normally. The Mn2+ activation was not associated with myosin light chain phosphorylation, in contrast to Ca2+-activated contractions. PMID:3186428

  8. Urine immunofixation electrophoresis remains important and is complementary to serum free light chain.

    PubMed

    Levinson, Stanley S

    2011-11-01

    Articles have debated whether or not urine analysis remains valuable for identifying monoclonal gammopathies. A general impression is that the newer serum free light chain (FLC) assay is more analytically sensitive, more quantitative and simpler to perform. Many laboratory directors may have seized on the idea of eliminating urine analysis because it is a tedious procedure and requires expert interpretation while most laboratories can perform automated serum FLC assay. Others have concluded that urine immunofixation electrophoresis (IFE) optimizes the diagnostic sensitivity and should be included when there is a clinical indication. Here, I show that papers faulting urine analysis often used inappropriate urine methodology and this helps explain why there was misinterpretation. Moreover, the literature, shows urine IFE is often more sensitive for identifying low-level monoclonal FLC than the serum assay because urine IFE is as sensitive when performed appropriately and generally more specific. Besides, the reference range for serum FLC assay is unclear which is a great problem in assessing response to treatment and in identifying diseases when there is low concentration monoclonal FLC. I conclude that urine IFE remains important and is complementary to serum FLC assay, although the best algorithms for use remains to be elucidated.

  9. Burden of cytogenetically abnormal plasma cells in light chain amyloidosis and their prognostic relevance.

    PubMed

    Kim, Seon Young; Im, Kyongok; Park, Si Nae; Kim, Jung-Ah; Yoon, Sung-Soo; Lee, Dong Soon

    2016-05-01

    We performed cytoplasmic fluorescence in situ hybridization assays of light chain amyloidosis (AL). In total, 234 patients were enrolled: 28 patients with AL, 24 with monoclonal gammopathy of undetermined significance (MGUS), and 182 with multiple myeloma (MM). Chromosomal abnormalities were detected in 13 of 22 (59%) AL patients without MM. All 13 patients demonstrated IGH rearrangement, and t(11;14)/IGH-CCND1 was most frequent (32%). Chromosome gain was not observed in AL patients without MM. These findings were dissimilar to findings in MGUS patients, in whom trisomy 9 was the most frequent abnormality. Of 6 AL patients with MM, 5 (83%) patients had cytogenetic abnormalities: 1q gain (4/6, 67%), gains of chromosome 9 (3/6, 50%), IGH rearrangement and RB1 (13q) deletions (2/6 each, 33%). The percentage of clonal plasma cells among total plasma cells was variable (median, 75%; range, 16-100%) for AL patients without MM, which was lower than the results for MM patients (median 100%). The overall survival of AL patients without MM was not significantly different according to the presence of cytogenetic abnormalities (P=0.510). In summary, among Korean AL patients, IGH rearrangement was the most frequent cytogenetic abnormality and cytogenetic aberration patterns differ compared with MGUS and MM patients. PMID:27015231

  10. AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain.

    PubMed

    Thaiparambil, Jose T; Eggers, Carrie M; Marcus, Adam I

    2012-08-01

    The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine(172) phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine(19) phosphorylated MRLC (pMRLC(ser19)) and spindle pole-associated pMRLC(ser19) are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC(ser19) spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC(ser19) to control spindle orientation via regulation of actin cortex-astral microtubule attachments.

  11. Conventional Kinesin Holoenzymes Are Composed of Heavy and Light Chain Homodimers†

    PubMed Central

    DeBoer, Scott R.; You, YiMei; Szodorai, Anita; Kaminska, Agnieszka; Pigino, Gustavo; Nwabuisi, Evelyn; Wang, Bin; Estrada-Hernandez, Tatiana; Kins, Stefan; Brady, Scott T.; Morfini, Gerardo

    2009-01-01

    Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations. PMID:18361505

  12. Myosin light chain kinase accelerates vesicle endocytosis at the calyx of Held synapse.

    PubMed

    Yue, Hai-Yuan; Xu, Jianhua

    2014-01-01

    Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca(2+) and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca(2+)/calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca(2+) channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca(2+)/calmodulin.

  13. Developmental Expression of IL-2-Receptor Light Chain (CD25) in the Chicken Embryo

    PubMed Central

    Fedecka-Bruner, Barbara; Penninger, Josef; Vaigot, Pierre; Lehmann, Anne; Martínez-A., Carlos

    1991-01-01

    Thymocyte differentiation obeys the same fundamental principles in mammals as in avian species. This parallelism does not only affect the developmentally controlled acquisition of CD3, 4, 8, and TcR isotype expression, but also concerns CD25, the light chain of the interleukin-2 receptor (IL-2R). On chicken thymocytes, surface CD25, which is recognized by the monoclonal antibody INN Ch16, is first observed during day 11 of embryonic life, and peaks at day 14, when it is expressed by about one-third of all lymphoid cells. CD25 is found on subsets of all ,thymocyte populations as defined by TcRαβ, TcRγδ, 2, CD4, and CD8 expression, cortical or medullary localization, and is also present on a subset of intrathymic nurse-cell lymphocytes. These findings suggest phylogenetic conservation of the IL-2/IL-2R-triggered differentiation pathway previously described for mammalian species, thus under-lining its probable functional importance. PMID:1840381

  14. An outer arm dynein light chain acts in a conformational switch for flagellar motility

    PubMed Central

    Patel-King, Ramila S.

    2009-01-01

    A system distinct from the central pair–radial spoke complex was proposed to control outer arm dynein function in response to alterations in the mechanical state of the flagellum. In this study, we examine the role of a Chlamydomonas reinhardtii outer arm dynein light chain that associates with the motor domain of the γ heavy chain (HC). We demonstrate that expression of mutant forms of LC1 yield dominant-negative effects on swimming velocity, as the flagella continually beat out of phase and stall near or at the power/recovery stroke switchpoint. Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme. Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain. We propose that this γ HC–LC1–microtubule ternary complex functions as a conformational switch to control outer arm activity. PMID:19620633

  15. Dynein light chain regulates axonal trafficking and synaptic levels of Bassoon.

    PubMed

    Fejtova, Anna; Davydova, Daria; Bischof, Ferdinand; Lazarevic, Vesna; Altrock, Wilko D; Romorini, Stefano; Schöne, Cornelia; Zuschratter, Werner; Kreutz, Michael R; Garner, Craig C; Ziv, Noam E; Gundelfinger, Eckart D

    2009-04-20

    Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. In this study, we report the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. We demonstrate that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon-DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material.

  16. Dynein light chain interaction with the peroxisomal import docking complex modulates peroxisome biogenesis in yeast.

    PubMed

    Chang, Jinlan; Tower, Robert J; Lancaster, David L; Rachubinski, Richard A

    2013-10-15

    Dynein is a large macromolecular motor complex that moves cargo along microtubules. A motor-independent role for the light chain of dynein, Dyn2p, in peroxisome biology in Saccharomyces cerevisiae was suggested from its interaction with Pex14p, a component of the peroxisomal matrix protein import docking complex. Here we show that cells of the yeast Yarrowia lipolytica deleted for the gene encoding the homologue of Dyn2p are impaired in peroxisome function and biogenesis. These cells exhibit compromised growth on medium containing oleic acid as the carbon source, the metabolism of which requires functional peroxisomes. Their peroxisomes have abnormal morphology, atypical matrix protein localization, and an absence of proteolytic processing of the matrix enzyme thiolase, which normally occurs upon its import into the peroxisome. We also show physical and genetic interactions between Dyn2p and members of the docking complex, particularly Pex17p. Together, our results demonstrate a role for Dyn2p in the assembly of functional peroxisomes and provide evidence that Dyn2p acts in cooperation with the peroxisomal matrix protein import docking complex to effect optimal matrix protein import.

  17. The regulation of RhoGEF Lfc by dynein light chain Tctex-1

    NASA Astrophysics Data System (ADS)

    Balan, Marc

    Lfc is a guanine nucleotide exchange factor (GEF) that activates the small GTPase RhoA, and its GEF activity is tightly regulated through protein-protein interactions, phosphorylation, and cellular localization. Lfc is anchored to microtubules through its interaction with the dynein light chain Tctex-1, which results in inhibition of Lfc's GEF activity. Here we present a crystallographic structure of Tctex-1 in complex with Lfc with residues 143-155 of Lfc bound at the Tctex-1 dimer interface. Structural alignment of our structure with Tctex-1 in complex with the dynein intermediate chain (DIC) shows the binding site of the DIC peptide and Lfc substantially overlap. Biochemical evidence, NMR perturbations assays and intrinsic fluorescence provide structural validation and support an extension of the Lfc binding site to the andalpha;-helices that may accommodate additional contact points with Tctex-1. We postulate a potential mechanism for Lfcandrsquo;s recruitment to the microtubules through a tripartite complex with Tctex-1 and DIC.

  18. Structure and function of outer dynein arm intermediate and light chain complex

    PubMed Central

    Oda, Toshiyuki; Abe, Tatsuki; Yanagisawa, Haruaki; Kikkawa, Masahide

    2016-01-01

    The outer dynein arm (ODA) is a molecular complex that drives the beating motion of cilia/flagella. Chlamydomonas ODA is composed of three heavy chains (HCs), two ICs, and 11 light chains (LCs). Although the three-dimensional (3D) structure of the whole ODA complex has been investigated, the 3D configurations of the ICs and LCs are largely unknown. Here we identified the 3D positions of the two ICs and three LCs using cryo–electron tomography and structural labeling. We found that these ICs and LCs were all localized at the root of the outer-inner dynein (OID) linker, designated the ODA-Beak complex. Of interest, the coiled-coil domain of IC2 extended from the ODA-Beak to the outer surface of ODA. Furthermore, we investigated the molecular mechanisms of how the OID linker transmits signals to the ODA-Beak, by manipulating the interaction within the OID linker using a chemically induced dimerization system. We showed that the cross-linking of the OID linker strongly suppresses flagellar motility in vivo. These results suggest that the ICs and LCs of the ODA form the ODA-Beak, which may be involved in mechanosignaling from the OID linker to the HCs. PMID:26864626

  19. Dictyostelium discoideum myosin: Isolation and characterization of cDNAs encoding the essential light chain

    SciTech Connect

    Chisholm, R.L.; Rushforth, A.M.; Pollenz, R.S.; Kuczmarski, E.R.; Tafuri, S.R.

    1988-02-01

    The authors used an antibody specific for Dictyostelium discoideum myosin to screen a lambdagt11 cDNA expression library to obtain cDNA clones which encode the Dictyostelium essential myosin light chain (EMLC). The amino acid sequence predicted from the sequence of the cDNA clone showed 31.5% identity with the amino acid sequence of the chicken EMLC. Comparisons of the Dictyostelium EMLC, a nonmuscle cell type, with EMLC sequences from similar MLCs of skeletal- and smooth-muscle origin, showed distinct regions of homology. Much of the observed homology was localized to regions corresponding to consensus Ca/sup 2 +/-binding of E-F hand domains. Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the EMLC. Examination of the pattern of EMLC mRNA expression showed that a significant increase in EMLC message levels occurred during the first few hours of development, coinciding with increased actin expression and immediately preceding the period of maximal chemotactic activity.

  20. Regulatory Light Chain Mutations Associated with Cardiomyopathy Affect Myosin Mechanics and Kinetics

    PubMed Central

    Greenberg, Michael J.; Watt, James D.; Jones, Michelle; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R.

    2009-01-01

    The myosin regulatory light chain (RLC) wraps around the alpha helical neck region of myosin. This neck region has been proposed to act as a lever arm, amplifying small conformational changes in the myosin head to generate motion. The RLC serves an important structural role, supporting the myosin neck region and a modulatory role, tuning the kinetics of the actin myosin interaction. Given the importance of the RLC, it is not surprising that mutations of the RLC can lead to familial hypertrophic cardiomyopathy (FHC), the leading cause of sudden cardiac death in people under 30. Population studies identified two FHC mutations located near the cationic binding site of the RLC, R58Q and N47K. Although these mutations are close in sequence, they differ in clinical presentation and prognosis with R58Q showing a more severe phenotype. We examined the molecular based changes in myosin that are responsible for the disease phenotype by purifying myosin from transgenic mouse hearts expressing mutant myosins and examining actin filament sliding using the in vitro motility assay. We found that both R58Q and N47K showed reductions in force compared to the wild type that could result in compensatory hypertrophy. Furthermore, we observed a higher ATPase rate and an increased activation at submaximal calcium levels for the R58Q myosin that could lead to decreased efficiency and incomplete cardiac relaxation, potentially explaining the more severe phenotype for the R58Q mutation. PMID:18929571

  1. Interhead fluorescence energy transfer between probes attached to translationally equivalent sites on the regulatory light chains of scallop myosin.

    PubMed

    Chantler, P D; Tao, T

    1986-11-01

    Interhead fluorescence energy transfer studies between probes located at translationally equivalent sites on the two heads of scallop myosin indicates that the distance between such sites is no less than 50 A. Regulatory light chains, possessing either one (Mercenaria, chicken gizzard) or two (Loligo, rabbit skeletal) sulfhydryl groups, were modified either with 1,5-IAEDANS (N'-iodoacetyl-N'-(1-sulfo-5-n-naphthyl)ethylenediamine), as energy transfer donor, or with IAF (5-(iodoacetamido)fluorescein) or DABMI (4-dimethylaminophenylazophenyl-4'-maleimide), as energy transfer acceptor. The sulfhydryl groups on these light chains are located at different positions within the regulatory light-chain primary sequence; this enables one to probe a variety of locations, with respect to regulatory light-chain topology, on each myosin head. These independently modified regulatory light chains were added back to desensitized scallop myosin under a variety of conditions, including biphasic re-addition, the aim being to maximize the number of interhead energy transfer couples present. The efficiency of energy transfer was determined on the same samples by both steady-state and time-decay techniques. Results obtained by these two techniques were in good agreement with each other and indicated that the efficiency of energy transfer did not exceed 20% in any of the hybrids studied. Transfer efficiencies were invariant, irrespective of the presence or absence of MgATP, calcium or actin, either separately or in combination. Results using heavy meromyosin at low ionic strength were identical. It is shown that these results, in conjunction with the results of recent crosslinking studies performed on comparable myosin hybrids, may place certain restrictions on the configurations of the two heads of myosin.

  2. [Omega-3 and health].

    PubMed

    Herbaut, C

    2006-09-01

    N-3 PUFA (omega-3), and the n-6 PUFA (omega-6) are essential fatty acids. They must be absorbed by alimentation and play a very important role in the coagulation (inhibition of platelets aggregation) and in the inflammatory reaction (anti-inflammatory effects). Their effects have been studied in different sicknesses. In cardiovascular diseases, particularly in coronary diseases, studies demonstrated a decreased mortality in populations who eat an omega-3 rich diet or who take an omega-3 supplement. Among others, sudden death after myocardial infarction is decreased. In inflammatory diseases an effect seem to be found in some studies. In rheumatoid arthritis a decrease of different biological markers of inflammation and in some case a clinical improvement has been noticed. It may be the same in COPD. On the other hand, they seem not to give any protection against cancer in general. At this moment the recommendations for healthy people are to eat twice a week fat fish and to take omega-3 rich oils. For pathological cases, recommendations exist only for coronary disease: 1 g of fish oils : mixture of eicosapentaenoic and docosahexaenoic acids (EPA/DHA) should be given after a myocardial infarction. PMID:17091903

  3. Phylogenetic origins of antibody structure. II. Immunoglobulins in the primary immune response of the bullfrog, Rana catesbiana.

    PubMed

    Marchalonis, J; Edelman, G M

    1966-11-01

    The anuran amphibian, Rana catesbiana, has been found to possess at least two kinds of immunoglobulins corresponding to gammaG- and gammaM-classes. These classes have the same chain structures as those of their counterparts in higher animal species. Light chains of both immunoglobulins had molecular weights of 20,000. Heavy chains of the gammaM-class had molecular weights of 72,100; those of the gammaG-class had molecular weights of 53,600. The carbohydrate content of the gammaG-immunoglobulin was 2.1%, and that of the gammaM-protein was 10.8%. The amino acid compositions of the immunoglobulins were generally similar to those of mammalian immunoglobulins. After a single injection of phage antigen (f2), the order of appearance of phage-neutralizing activity in the frog immunoglobulin classes was (a) gammaM-antibodies, and (b) gammaG-antibodies. The results of this and previous studies suggest that the gammaG-immunoglobulins emerged at some point in evolution between the elasmobranchs and the anuran amphibians. PMID:4162734

  4. Rescue and expression of human immunoglobulin genes to generate functional human monoclonal antibodies.

    PubMed

    Lewis, A P; Parry, N; Peakman, T C; Crowe, J S

    1992-07-01

    Human monoclonal antibody production has been hampered for many years by the instability of cell lines and low levels of expression of the antibodies. We describe here the rescue of human immunoglobulin genes utilizing micro-mRNA preparation from a small number of human hybridoma cells and conventional cDNA cloning. This allows cloning and immediate high-level expression from full-length human heavy and light chain cDNA molecules and provides a mechanism to rescue whole human monoclonal antibodies of proven efficacy.

  5. From Alpha To Omega

    NASA Astrophysics Data System (ADS)

    Castellano, Doc

    2002-05-01

    Galileo, the Father of Modern Science put forth the first significant Modern Scientific Era/Philosophy. Best represented per: x' = x (+/-) vt. Locating/defining the dynamic x' per a fixed, Cartesian Coordinate, reference frame.----- Einstein, the popularized relativist, utilizing Lorentz's transformation Equations: x' = (x-vt)/squareroot [1 - (v squared/c squared)], c the velocity of light. Arbitrarily decreed that c must be the ultimate universal velocity. Thus, Reporters, the general Public, and Scientists consider/considered, Einstein's OPINION of our Universe, the 'Omega Concept'. ----- Castellano, since 1955, has PROVEN his "Castellano Transformation Equations": X' = (X - vt)/squareroot [ 1 - (v squared/c squared)]. Capital C = or greater than c; IS THE OMEGA CONCEPT. And his "MAPHICS" combining the Philosophy of Mathematics with the Philosophy of Physics is "THE OMEGA PHILOSOPHY". Sufficient PROOFS and details at: http://hometown.aol.com/phdco/myhomepage/index.html Thank you for your interest. My sincere appreciation for your attention and deserved acknowledgments.

  6. From Alpha To Omega

    NASA Astrophysics Data System (ADS)

    Castellano, Doc

    2002-08-01

    Galileo, the Father of Modern Science, put forth the first significant Modern Scientific Era/Philosophy. Best represented per: x' = x (+/-) vt. Locating/defining the dynamic x' in an Euclidean, fixed frame Universe. Einstein, the popularized relativist, utilizing Lorentz's transformation equations: x' = (x - vt)/square root [ 1- (v squared/c squared)], c the velocity of light. Arbitrarily decreed that c must be the ultimate, universal velocity. Thus, Reporters, the general Public and Scientists consider/considered, Einstein's OPINION of our Universe, 'The Omega Concept'. Castellano, since 1954, has PROVEN the "C Transformation Equations": X' = (X - vt)/square root [ 1 - (v squared/C squared)], Capital C = or greater than c; IS THE OMEGA CONCEPT. And "MAPHICS", combining the Philosophy of Mathematics with the Philosophy of Physics is "THE OMEGA PHILOSOPHY". Sufficient PROOFS & details are at: http://hometown.aol.com/phdco/myhomepage/index/html ----- Thank you for your interest. My sincere appreciation for deserved acknowledgements.

  7. Microinjection of antibodies to the calpactin I light chain in MDBK cells causes precipition of the cytoskeletal calpactin I complex without affecting the distribution of related proteins.

    PubMed

    Glenney, J R

    1990-01-01

    The calpactin I complex is composed of two heavy chain (39K) and two light chain (11K) subunits. The heavy chain is a member of a protein family that includes lipocortins, endonexin and chromobindins while the light chain is a member of the S100 family (7 distinct members are known). We have found that the kidney epithelial cell line MDBK expresses four members of the heavy chain family and two members of the light chain protein family. Antibodies to the light chain of calpactin I were found to cause the precipitation of injected antibody together with the associated heavy chain without apparent effect on the distribution of related proteins. This suggests a differential targeting of various members of the calpactin heavy and light chain families even within the same cell.

  8. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load.

    PubMed

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R

    2015-08-15

    Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31-41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level. PMID:26116789

  9. Elevated neurofilament light chain (NFL) mRNA levels in prediabetic peripheral neuropathy.

    PubMed

    Celikbilek, Asuman; Tanik, Nermin; Sabah, Seda; Borekci, Elif; Akyol, Lutfi; Ak, Hakan; Adam, Mehmet; Suher, Murat; Yilmaz, Neziha

    2014-06-01

    Evidence suggests that peripheral nerve injury occurs during the early stages of disease with mild glycemic dysregulation. Two proteins, neuron-specific enolase (NSE) and neurofilament light chain (NFL), have been examined previously as possible markers of neuronal damage in the pathophysiology of neuropathies. Herein, we aimed to determine the potential value of circulatory NSE and NFL mRNA levels in prediabetic patients and in those with peripheral neuropathy. This prospective clinical study included 45 prediabetic patients and 30 age- and sex-matched controls. All prediabetic patients were assessed with respect to diabetes-related microvascular complications, such as peripheral neuropathy, retinopathy and nephropathy. mRNA levels of NSE and NFL were determined in the blood by real-time polymerase chain reaction. NSE mRNA levels were similar between prediabetic and control groups (p > 0.05), whereas NFL mRNA levels were significantly higher in prediabetics than in controls (p < 0.001). NSE mRNA levels did not significantly differ between prediabetic patients with and without peripheral neuropathy (p > 0.05), while NFL mRNA levels were significantly higher in prediabetics with peripheral neuropathy than in those without (p = 0.038). According to correlation analysis, NFL mRNA levels were positively correlated with the Douleur Neuropathique 4 questionnaire score in prediabetic patients (r = 0.302, p = 0.044). This is the first study to suggest blood NFL mRNA as a surrogate marker for early prediction of prediabetic peripheral neuropathy, while NSE mRNA levels may be of no diagnostic value in prediabetic patients.

  10. Regulatory light chain mutants linked to heart disease modify the cardiac myosin lever arm.

    PubMed

    Burghardt, Thomas P; Sikkink, Laura A

    2013-02-19

    Myosin is the chemomechanical energy transducer in striated heart muscle. The myosin cross-bridge applies impulsive force to actin while consuming ATP chemical energy to propel myosin thick filaments relative to actin thin filaments in the fiber. Transduction begins with ATP hydrolysis in the cross-bridge driving rotary movement of a lever arm converting torque into linear displacement. Myosin regulatory light chain (RLC) binds to the lever arm and modifies its ability to translate actin. Gene sequencing implicated several RLC mutations in heart disease, and three of them are investigated here using photoactivatable GFP-tagged RLC (RLC-PAGFP) exchanged into permeabilized papillary muscle fibers. A single-lever arm probe orientation is detected in the crowded environment of the muscle fiber by using RLC-PAGFP with dipole orientation deduced from the three-spatial dimension fluorescence emission pattern of the single molecule. Symmetry and selection rules locate dipoles in their half-sarcomere, identify those at the minimal free energy, and specify active dipole contraction intermediates. Experiments were performed in a microfluidic chamber designed for isometric contraction, total internal reflection fluorescence detection, and two-photon excitation second harmonic generation to evaluate sarcomere length. The RLC-PAGFP reports apparently discretized lever arm orientation intermediates in active isometric fibers that on average produce the stall force. Disease-linked mutants introduced into RLC move intermediate occupancy further down the free energy gradient, implying lever arms rotate more to reach stall force because mutant RLC increases lever arm shear strain. A lower free energy intermediate occupancy involves a lower energy conversion efficiency in the fiber relating a specific myosin function modification to the disease-implicated mutant.

  11. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

    SciTech Connect

    Muthu, Priya; Wang, Li; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Huang, Wenrui; Hernandez, Olga M.; Kawai, Masataka; Irving, Thomas C.; Szczesna-Cordary, Danuta

    2012-04-02

    The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-{Delta}43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing ({approx} 1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I{sub 1,1}/I{sub 1,0}, indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-{Delta}43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-{Delta}43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.

  12. Early Reduction of Serum-Free Light Chains Associates with Renal Recovery in Myeloma Kidney

    PubMed Central

    Cockwell, Paul; Stringer, Stephanie; Bradwell, Arthur; Cook, Mark; Gertz, Morie A.; Dispenzieri, Angela; Winters, Jeffrey L.; Kumar, Shaji; Rajkumar, S. Vincent; Kyle, Robert A.; Leung, Nelson

    2011-01-01

    Myeloma kidney is the major cause of severe irreversible renal failure in patients with multiple myeloma. This tubulointerstitial injury is a direct consequence of high concentrations of circulating monoclonal free light chains (FLCs) produced by a clonal expansion of plasma cells. Early reduction of serum FLCs associates with renal recovery, but the target threshold of reduction to facilitate renal recovery is unknown. To determine the relationship between the achieved FLC reduction and renal recovery, we identified 39 patients with biopsy-proven myeloma kidney, the majority of whom had severe renal failure at presentation (median estimated GFR 9 ml/min per 1.73 m2). In a multivariable analysis incorporating demographic, hematologic, and renal variables, only the achieved FLC reduction significantly predicted renal recovery (P = 0.003). The relationship between renal recovery and FLC reduction was linear with no absolute threshold for FLC reduction. A 60% reduction in FLCs by day 21 associated with recovery of renal function for 80% of the population. Patient survival strongly associated with renal recovery: the median survival was 42.7 months (range 0 to 80) among those who recovered function compared with 7.8 months (range 0 to 54) among those who did not (P < 0.02). Cox-regression analysis demonstrated that the first presentation of myeloma, the kappa isotype of FLC, and renal recovery were independent predictors of survival. In conclusion, recovery of renal function in myeloma kidney depends on early reduction of serum FLCs, and this recovery associates with a significant survival advantage. PMID:21511832

  13. Constraints on intron evolution in the gene encoding the myosin alkali light chain in Drosophila

    SciTech Connect

    Leicht, B.G.; Muse, S.V.; Hanczyc, M.

    1995-01-01

    Interspecific comparisons of intron sequences reveal conserved blocks of invariant nucleotides and several other departures from the strictly neutral model of molecular evolution. To distinguish the past action of evolutionary forces in introns known to have regulatory information, we examined nucleotide sequence variation at 991 sites in a random sample of 16 Drosophila melanogaster alleles of the gene encoding the myosin alkali light chain (Mlc1). The Mlc1 gene of D. melanogaster encodes two Mlc1 isoforms via developmentally regulated alternative pre-mRNA splicing. Analyses of these data reveal that introns 4 and 5, which flank the alternatively spliced exon 5, have reduced levels of both intraspecific polymorphism and interspecific divergence relative to intron 3. No polymorphism was observed in any of the exons examined in D. melanogaster. A genealogical analysis clearly demonstrates the occurrence of intragenic recombination in the ancestral history of Mlc1. Recombination events are estimated to be 13 times more likely than mutation events over the span of the sequenced region. Although there is little evidence for pairwise linkage disequilibrium in the Mlc1 region, higher order disequilibrium. does seem to be present in the 5{prime} half of the portion of the gene that was examined. Predictions of the folding free energy of the pre-mRNA reveal that sampled alleles have a significantly higher (less stable) free energy than do randomly permuted sequences. These results are consistent with the hypothesis that introns surrounding an alternatively spliced exon are subjected to additional constraints, perhaps due to specific aspects of secondary structure required for appropriate splicing of the pre-mRNA molecule. 48 refs., 5 figs., 3 tabs.

  14. [Role of phosphatidylinositol 3-kinase and myosin light chain kinase during the activation of thrombin receptors].

    PubMed

    Han, Yue; Gao, Hai-Li; Zhang, Wei; Bai, Xia; Dai, Lan; Sheng, Wen-Hong; Sun, Ai-Ning; Wu, De-Pei; Wang, Zhao-Yue; Ruan, Chang-Geng

    2009-06-01

    The objective of study was to compare the influences of wortmannin on platelet aggregation and platelet membrane surface glycoproteins GPIb expression after thrombin receptor activation, and to investigate the role of phosphatidylinositol 3-kinase (PI3-K) and myosin light chain kinase (MLCK) in the course of thrombin receptor activation. Peptide SFLLRN (PAR1-AP) and AYPGKF (PAR4-AP) were used for stimulating platelet, and the changes of platelet aggregation and GPIb were analyzed with 100 nmol/L wortmannin (inhibitor of PI3-K) and 10 micromol/L wortmannin (inhibitor of MLCK). The results indicated that the platelet activation was influenced by either concentration of wortmannin in response to PAR stimulation. Platelet aggregation was apparently inhibited by 10 micromol/L wortmannin through both PAR peptides, and was slightly inhibited by 100 nmol/L wortmannin only under PAR1-AP activation. In addition, GPIbalpha internalization was partly inhibited by 100 nmol/L wortmannin in response to PAR1 (p < 0.05 at 1, 2, 5 min) and PAR4 (p < 0.05 at 2, 5, 10 min) activation. Meanwhile, 10 micromol/L wortmannin induced little change for GPIbalpha centralisation in the course of PAR activation, with a delayed restoration of surface GPIbalpha observed under PAR1-AP activation, and no change of GPIbalpha redistribution existed under PAR4-AP activation. It is concluded that the different roles of PI3-K and MLCK exist in the course of thrombin receptor activation. PI3-K accelerates the short course of GPIb centralisation for two PAR signal pathways, while MLCK inhibits the restoration of GPIbalpha in PAR1 pathway. PMID:19549383

  15. Myosin light chain kinase controls voltage-dependent calcium channels in vascular smooth muscle.

    PubMed

    Martinsen, A; Schakman, O; Yerna, X; Dessy, C; Morel, N

    2014-07-01

    The Ca(2+)-dependent kinase myosin light chain kinase (MLCK) is the activator of smooth muscle contraction. In addition, it has been reported to be involved in Ca(2+) channel regulation in cultured cells, and we previously showed that the MLCK inhibitor ML-7 decreases arginine vasopressin (AVP)-induced Ca(2+) influx in rat aorta. This study was designed to investigate whether MLCK is involved in Ca(2+) regulation in resistance artery smooth muscle cell, which plays a major role in the control of blood pressure. As ML compounds were shown to have off-target effects, MLCK was downregulated by transfection with a small interfering RNA targeting MLCK (MLCK-siRNA) in rat small resistance mesenteric artery (RMA) and in the rat embryonic aortic cell line A7r5. Noradrenaline-induced contraction and Ca(2+) signal were significantly depressed in MLCK-siRNA compared to scramble-siRNA-transfected RMA. Contraction and Ca(2+) signal induced by high KCl and voltage-activated Ca(2+) current were also significantly decreased in MLCK-siRNA-transfected RMA, suggesting that MLCK depletion modifies voltage-operated Ca(2+) channels. KCl- and AVP-induced Ca(2+) signals and voltage-activated Ca(2+) current were decreased in MLCK-depleted A7r5 cells. Eventually, real-time quantitative PCR analysis indicated that in A7r5, MLCK controlled mRNA expression of CaV1.2 (L-type) and CaV3.1 (T-type) voltage-dependent Ca(2+) channels. Our results suggest that MLCK controls the transcription of voltage-dependent Ca(2+) channels in vascular smooth muscle cells. PMID:24162233

  16. Comparing domain interactions within antibody Fabs with kappa and lambda light chains

    PubMed Central

    Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W.; Dickey, Mark; Froning, Karen; Conner, Elaine M.; Cujec, Thomas P.; Demarest, Stephen J.

    2016-01-01

    ABSTRACT IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates. PMID:27454112

  17. Factor VII Light Chain-Targeted Lidamycin Shows Intensified Therapeutic Efficacy for Liver Cancer

    PubMed Central

    Liu, Xiujun; Xu, Shuangshuang; Li, Caihong; Zhang, Yang; Yang, Jie; Zheng, Junnian

    2012-01-01

    Abstract The overexpression of tissue factor (TF) observed in numerous cancer cells and clinical samples of human cancers makes TF an ideal target for cancer therapy. The purpose of this study is to develop a TF-targeting energized fusion protein hlFVII-LDP-AE, which is composed of a human Factor VII light chain (hlFVII) as the targeting domain conjugated to the cytotoxic antibiotic lidamycin (LDM, LDP-AE) as the effector domain. The potential efficacy of hlFVII-LDP-AE for cancer therapy was tested in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays and in vivo with a BALB/c nude mouse xenograft model of human liver cancer line HepG2. The inhibitory concentration (IC50) value of hlFVII-LDP-AE varied from 0.15 to 0.64 nM for the various human tumor lines. hlFVII-LDP-AE showed a tumor growth inhibition rate of 90.6% at the dose of 0.6 mg/kg in in vivo animal experiments. The mechanism through which hlFVII-LDP-AE inhibits tumor growth also was determined by Hoechst 33342 staining and Tdt-mediated dUTP nick-end labeling (TUNEL) assay. hlFVII-LDP-AE causes tumor cell death through inducing chromatin condensation and cleavage of genomic DNA. These findings suggest that the hlFVII-LDP-AE protocol is efficacious and tolerated in the mouse model of human liver cancer HepG2 and has clinical applicability for treating cancer patients. PMID:22651685

  18. High accuracy OMEGA timekeeping

    NASA Technical Reports Server (NTRS)

    Imbier, E. A.

    1982-01-01

    The Smithsonian Astrophysical Observatory (SAO) operates a worldwide satellite tracking network which uses a combination of OMEGA as a frequency reference, dual timing channels, and portable clock comparisons to maintain accurate epoch time. Propagational charts from the U.S. Coast Guard OMEGA monitor program minimize diurnal and seasonal effects. Daily phase value publications of the U.S. Naval Observatory provide corrections to the field collected timing data to produce an averaged time line comprised of straight line segments called a time history file (station clock minus UTC). Depending upon clock location, reduced time data accuracies of between two and eight microseconds are typical.

  19. Structure of the Single-lobe Myosin Light Chain C in Complex with the Light Chain-binding Domains of Myosin-1C Provides Insights into Divergent IQ Motif Recognition.

    PubMed

    Langelaan, David N; Liburd, Janine; Yang, Yidai; Miller, Emily; Chitayat, Seth; Crawley, Scott W; Côté, Graham P; Smith, Steven P

    2016-09-01

    Myosin light chains are key regulators of class 1 myosins and typically comprise two domains, with calmodulin being the archetypal example. They bind IQ motifs within the myosin neck region and amplify conformational changes in the motor domain. A single lobe light chain, myosin light chain C (MlcC), was recently identified and shown to specifically bind to two sequentially divergent IQ motifs of the Dictyostelium myosin-1C. To provide a molecular basis of this interaction, the structures of apo-MlcC and a 2:1 MlcC·myosin-1C neck complex were determined. The two non-functional EF-hand motifs of MlcC pack together to form a globular four-helix bundle that opens up to expose a central hydrophobic groove, which interacts with the N-terminal portion of the divergent IQ1 and IQ2 motifs. The N- and C-terminal regions of MlcC make critical contacts that contribute to its specific interactions with the myosin-1C divergent IQ motifs, which are contacts that deviate from the traditional mode of calmodulin-IQ recognition.

  20. New Light Chain Amyloid Response Criteria Help Risk Stratification of Patients by Day 100 after Autologous Hematopoietic Cell Transplantation.

    PubMed

    D'Souza, Anita; Huang, Jiaxing; Hari, Parameswaran

    2016-04-01

    Hematologic response criteria in light chain (AL) amyloidosis were updated in 2012 to incorporate free light chain responses. These criteria have been validated in autologous hematopoietic cell transplantation in AL at 6 and 12 months after transplantation. Using a transplantation registry, we assessed day 100 responses in AL amyloidosis. We validate the prognostic significance of the new criteria at this time point. Further, we show that patients who do not achieve at least a very good partial response by this time point have equally worse outcomes, regardless of depth of response (partial versus no response). Thus, we conclude that the new criteria help identify the poor responders by day 100 after transplantation and that this subset of patients should be studied for early evaluation in consolidation trials.

  1. Rearrangements of chicken immunoglobulin genes in lymphoid cells transformed by the avian retroviral oncogene v-rel.

    PubMed

    Chen, L; Lim, M Y; Bose, H; Bishop, J M

    1988-01-01

    The retroviral oncogene v-rel transforms poorly characterized lymphoid cells. We have explored the nature of these cells by analyzing the configuration and expression of immunoglobulin genes in chicken hemopoietic cells transformed by v-rel. None of the transformed cells expressed their immunoglobulin genes. The cells fell into three classes: class I cells have their immunoglobulin genes potentially in an embryonic configuration; class II and class III cells have lost one copy of the lambda light chain locus and have one copy of the heavy chain locus rearranged into a configuration that differs from what is found in mature B cells. In class II cells, the other heavy chain locus may be in embryonic configuration, whereas it is deleted in class III cells. The first of these classes may represent the earliest stage of the lymphoid lineage yet encountered among virus-transformed cells, whereas the second and third classes represent an apparently anomalous rearrangement whose origin remains unknown.

  2. Omega-AB

    SciTech Connect

    Siirola, John D.; Slepoy, Alexander; Sprigg, Jr., James A.; Jorgensen, Craig R.; Selzler, Gene; Pryor, Richard J.

    2007-05-01

    A hierarchical, modular modeling environment for hybrid simulations of sequential-modular, systems dynamics, discrete-event, and agent-based paradigms Omega-AB models contain a hierarchically-defined module tree that specifies the execution logic for the simulation, and a multi-network graph that defines the environment within which the simulation occurs. Modules are the fundamental buildinig blocks of an Omega-AB model and can define anything from a basic mathematical operation to a complex behavioral response model. Modules rely on the "plug-in" concept which allows developers to build independent module libraries that are gathered, linked, and instantiated by the Omega-AB engine at run time. Inter-module communication occurs through two complimentary systems: pull-based "ports" for general computation patterns and push-based "plugs" for event processing. The simulation environment is an abstract graph of nodes and links. Agents (module sub-trees headed up by an Agent module) reside at nodes and relate to their neighbors through typed links. To facilitate the construction and visualization of complex, interacting networks with dramatically different structure, Omega-AB provides a system for organizing the nodes into hierarchica trees that describe "slices" of the overall network.

  3. Simplified OMEGA receivers

    NASA Technical Reports Server (NTRS)

    Burhans, R. W.

    1974-01-01

    The details are presented of methods for providing OMEGA navigational information including the receiver problem at the antenna and informational display and housekeeping systems based on some 4 bit data processing concepts. Topics discussed include the problem of limiters, zero crossing detectors, signal envelopes, internal timing circuits, phase counters, lane position displays, signal integrators, and software mapping problems.

  4. Omega-AB

    2007-05-01

    A hierarchical, modular modeling environment for hybrid simulations of sequential-modular, systems dynamics, discrete-event, and agent-based paradigms Omega-AB models contain a hierarchically-defined module tree that specifies the execution logic for the simulation, and a multi-network graph that defines the environment within which the simulation occurs. Modules are the fundamental buildinig blocks of an Omega-AB model and can define anything from a basic mathematical operation to a complex behavioral response model. Modules rely on the "plug-in" conceptmore » which allows developers to build independent module libraries that are gathered, linked, and instantiated by the Omega-AB engine at run time. Inter-module communication occurs through two complimentary systems: pull-based "ports" for general computation patterns and push-based "plugs" for event processing. The simulation environment is an abstract graph of nodes and links. Agents (module sub-trees headed up by an Agent module) reside at nodes and relate to their neighbors through typed links. To facilitate the construction and visualization of complex, interacting networks with dramatically different structure, Omega-AB provides a system for organizing the nodes into hierarchica trees that describe "slices" of the overall network.« less

  5. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein

    PubMed Central

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

  6. Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

    PubMed Central

    Guhathakurta, Piyali; Prochniewicz, Ewa; Thomas, David D.

    2015-01-01

    We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40–45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC’s location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders. PMID:25825773

  7. Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain.

    PubMed

    Guhathakurta, Piyali; Prochniewicz, Ewa; Thomas, David D

    2015-04-14

    We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40-45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC's location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders. PMID:25825773

  8. Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains*

    PubMed Central

    Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.; Sánchez-Alcalá, Rosalba; Olamendi-Portugal, Timoteo; Rudiño-Piñera, Enrique; Rojas, Sonia; Sánchez-López, Rosana; Fernández-Velasco, Daniel A.; Becerril, Baltazar

    2015-01-01

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL. PMID:25505244

  9. [Avidity of polyreactive immunoglobulins].

    PubMed

    Bobrovnik, S A

    2014-01-01

    An analysis of the mechanism of interaction between polyreactive immunoglobulins (PRIG) and antigen was conducted and it was shown that most of the traditional methods of antibody affinity evaluation are not applicable for PRIG affinity. The comparative assessment of the mouse and human PRIG avidity against ovalbumin and horse myoglobin and the avidity of specific monoclonal antibodies against ovalbumin have shown that the avidity of PRIG not only is much less than the avidity of monoclonal antibodies but even exceeds it.

  10. CaMKII and at least two unidentified kinases phosphorylate regulatory light chain in non-contracting cardiomyocytes.

    PubMed

    Eikemo, Hilde; Moltzau, Lise Román; Nguyen, Cam H T; Levy, Finn Olav; Skomedal, Tor; Osnes, Jan-Bjørn

    2016-08-12

    In cardiac tissue, regulatory light chain (RLC, myosin light chain 2) phosphorylation (Ser(15)) leads to modulation of muscle contraction through Ca(2+)-sensitization. To elucidate which kinases that are involved in the basal (diastolic phase) RLC phosphorylation, we studied non-contracting adult rat cardiomyocytes. RLC kinase activities in situ were unmasked by maximally inhibiting myosin light chain phosphatase (MLCP) by calyculin A in the absence and presence of various protein kinase inhibitors. Surprisingly MLCK did not contribute to the phosphorylation of RLC in the non-contracting cardiomyocytes. Two kinase activity groups were revealed by different sensitivities to staurosporine. The fraction with the highest sensitivity to staurosporine was inhibited by KN-93, a selective CaMKII inhibitor, producing a 23% ± 7% reduction in RLC phosphorylation. Calmodulin antagonism (W7) and reduction in Ca(2+) (EGTA) combined with low concentration of staurosporine caused a larger decrease in RLC phosphorylation than staurosporine alone. These data strongly suggest that in addition to CaMKII, there is another Ca(2+)/calmodulin-dependent kinase and a Ca(2+)/calmodulin-independent kinase phosphorylating RLC. Thus the RLC phosphorylation seems to be ensured by redundant kinase activities.

  11. TCTEX1D4 Interactome in Human Testis: Unraveling the Function of Dynein Light Chain in Spermatozoa

    PubMed Central

    Freitas, Maria João; Korrodi-Gregório, Luís; Morais-Santos, Filipa; da Cruz e Silva, Edgar

    2014-01-01

    Abstract Studies were designed to identify the TCTEX1D4 interactome in human testis, with the purpose of unraveling putative protein complexes essential to male reproduction and thus novel TCTEX1D4 functions. TCTEX1D4 is a dynein light chain that belongs to the DYNT1/TCTEX1 family. In spermatozoa, it appears to be important to sperm motility, intraflagellar transport, and acrosome reaction. To contribute to the knowledge on TCTEX1D4 function in testis and spermatozoa, a yeast two-hybrid assay was performed in testis, which allowed the identification of 40 novel TCTEX1D4 interactors. Curiously, another dynein light chain, TCTEX1D2, was identified and its existence demonstrated for the first time in human spermatozoa. Immunofluorescence studies proved that TCTEX1D2 is an intra-acrosomal protein also present in the midpiece, suggesting a role in cargo movement in human spermatozoa. Further, an in silico profile of TCTEX1D4 revealed that most TCTEX1D4 interacting proteins were not previously characterized and the ones described present a very broad nature. This reinforces TCTEX1D4 as a dynein light chain that is capable of interacting with a variety of functionally different proteins. These observations collectively contribute to a deeper molecular understanding of the human spermatozoa function. PMID:24606217

  12. Myosin light chain kinase steady-state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates

    PubMed Central

    Alcala, Diego B.; Haldeman, Brian D.; Brizendine, Richard K.; Krenc, Agata K.; Baker, Josh E.; Rock, Ronald S.; Cremo, Christine R.

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat/Km) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. PMID:27528075

  13. TCTEX1D4 interactome in human testis: unraveling the function of dynein light chain in spermatozoa.

    PubMed

    Freitas, Maria João; Korrodi-Gregório, Luís; Morais-Santos, Filipa; Cruz e Silva, Edgar da; Fardilha, Margarida

    2014-04-01

    Studies were designed to identify the TCTEX1D4 interactome in human testis, with the purpose of unraveling putative protein complexes essential to male reproduction and thus novel TCTEX1D4 functions. TCTEX1D4 is a dynein light chain that belongs to the DYNT1/TCTEX1 family. In spermatozoa, it appears to be important to sperm motility, intraflagellar transport, and acrosome reaction. To contribute to the knowledge on TCTEX1D4 function in testis and spermatozoa, a yeast two-hybrid assay was performed in testis, which allowed the identification of 40 novel TCTEX1D4 interactors. Curiously, another dynein light chain, TCTEX1D2, was identified and its existence demonstrated for the first time in human spermatozoa. Immunofluorescence studies proved that TCTEX1D2 is an intra-acrosomal protein also present in the midpiece, suggesting a role in cargo movement in human spermatozoa. Further, an in silico profile of TCTEX1D4 revealed that most TCTEX1D4 interacting proteins were not previously characterized and the ones described present a very broad nature. This reinforces TCTEX1D4 as a dynein light chain that is capable of interacting with a variety of functionally different proteins. These observations collectively contribute to a deeper molecular understanding of the human spermatozoa function.

  14. Immunoglobulin genes of the turtles.

    PubMed

    Magadán-Mompó, Susana; Sánchez-Espinel, Christian; Gambón-Deza, Francisco

    2013-03-01

    The availability of reptile genomes for the use of the scientific community is an exceptional opportunity to study the evolution of immunoglobulin genes. The genome of Chrysemys picta bellii and Pelodiscus sinensis is the first one that has been reported for turtles. The scanning for immunoglobulin genes resulted in the presence of a complex locus for the immunoglobulin heavy chain (IGH). This IGH locus in both turtles contains genes for 13 isotypes in C. picta bellii and 17 in P. sinensis. These correspond with one immunoglobulin M, one immunoglobulin D, several immunoglobulins Y (six in C. picta bellii and eight in P. sinensis), and several immunoglobulins that are similar to immunoglobulin D2 (five in C. picta belli and seven in P. sinensis) that was previously described in Eublepharis macularius. It is worthy to note that IGHD2 are placed in an inverted transcriptional orientation and present sequences for two immunoglobulin domains that are similar to bird IgA domains. Furthermore, its phylogenetic analysis allows us to consider about the presence of IGHA gene in a primitive reptile, so we would be dealing with the memory of the gene that originated from the bird IGHA. In summary, we provide a clear picture of the immunoglobulins present in a turtle, whose analysis supports the idea that turtles emerged from the evolutionary line from the differentiation of birds and the presence of the IGHA gene present in a common ancestor.

  15. Studies on immunoglobulin gene rearrangement in formalin-fixed, paraffin-embedded pathology specimens.

    PubMed

    Dubeau, L; Weinberg, K; Jones, P A; Nichols, P W

    1988-03-01

    Studies on immunoglobulin gene rearrangement in lymphoid lesions are an increasingly important application of molecular biology in diagnostic medicine. The authors have therefore examined the possibility of detecting such rearrangements in formalin-fixed, paraffin-embedded pathology specimens. Southern blots of DNA obtained from optimally fixed tissues were very similar to blots of unfixed material, except that the electrophoretic mobility of the fixed DNA fragments was sometimes slightly reduced. High-molecular-weight DNA was not recovered from suboptimally fixed partially autolysed samples. Increasing the time of exposure to formalin resulted in loss of hybridizable DNA. Monoclonal rearrangements of heavy and light chain immunoglobulin genes could be detected in formalin-fixed specimens provided that these fixation artifacts were taken into consideration. This technique expands the pool of material available for studies on gene rearrangement and should facilitate the use of such studies in clinical medicine.

  16. Immunoglobulin genes and diversity: what we have learned from domestic animals.

    PubMed

    Sun, Yi; Liu, Zhancai; Ren, Liming; Wei, Zhiguo; Wang, Ping; Li, Ning; Zhao, Yaofeng

    2012-06-20

    This review focuses on the diversity of immunoglobulin (Ig) genes and Ig isotypes that are expressed in domestic animals. Four livestock species-cattle, sheep, pigs, and horses-express a full range of Ig heavy chains (IgHs), including μ, δ, γ, ϵ, and α. Two poultry species (chickens and ducks) express three IgH isotypes, μ, υ, and α, but not δ. The κ and λ light chains are both utilized in the four livestock species, but only the λ chain is expressed in poultry. V(D)J recombination, somatic hypermutation (SHM), and gene conversion (GC) are three distinct mechanisms by which immunoglobulin variable region diversity is generated. Different domestic animals may use distinct means to diversify rearranged variable regions of Ig genes.

  17. Enhanced paracellular transport of insulin can be achieved via transient induction of myosin light chain phosphorylation.

    PubMed

    Taverner, Alistair; Dondi, Ruggero; Almansour, Khaled; Laurent, Floriane; Owens, Siân-Eleri; Eggleston, Ian M; Fotaki, Nikoletta; Mrsny, Randall J

    2015-07-28

    The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein-protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4kDa fluorescent dextran but not 70kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3-4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the intestinal

  18. Serum neurofilament light chain protein is a measure of disease intensity in frontotemporal dementia

    PubMed Central

    Woollacott, Ione O.C.; Dick, Katrina M.; Brotherhood, Emilie; Gordon, Elizabeth; Fellows, Alexander; Toombs, Jamie; Druyeh, Ronald; Cardoso, M. Jorge; Ourselin, Sebastien; Nicholas, Jennifer M.; Norgren, Niklas; Mead, Simon; Andreasson, Ulf; Blennow, Kaj; Schott, Jonathan M.; Fox, Nick C.; Warren, Jason D.; Zetterberg, Henrik

    2016-01-01

    Objective: To investigate serum neurofilament light chain (NfL) concentrations in frontotemporal dementia (FTD) and to see whether they are associated with the severity of disease. Methods: Serum samples were collected from 74 participants (34 with behavioral variant FTD [bvFTD], 3 with FTD and motor neuron disease and 37 with primary progressive aphasia [PPA]) and 28 healthy controls. Twenty-four of the FTD participants carried a pathogenic mutation in C9orf72 (9), microtubule-associated protein tau (MAPT; 11), or progranulin (GRN; 4). Serum NfL concentrations were determined with the NF-Light kit transferred onto the single-molecule array platform and compared between FTD and healthy controls and between the FTD clinical and genetic subtypes. We also assessed the relationship between NfL concentrations and measures of cognition and brain volume. Results: Serum NfL concentrations were higher in patients with FTD overall (mean 77.9 pg/mL [SD 51.3 pg/mL]) than controls (19.6 pg/mL [SD 8.2 pg/mL]; p < 0.001). Concentrations were also significantly higher in bvFTD (57.8 pg/mL [SD 33.1 pg/mL]) and both the semantic and nonfluent variants of PPA (95.9 and 82.5 pg/mL [SD 33.0 and 33.8 pg/mL], respectively) compared with controls and in semantic variant PPA compared with logopenic variant PPA. Concentrations were significantly higher than controls in both the C9orf72 and MAPT subgroups (79.2 and 40.5 pg/mL [SD 48.2 and 20.9 pg/mL], respectively) with a trend to a higher level in the GRN subgroup (138.5 pg/mL [SD 103.3 pg/mL). However, there was variability within all groups. Serum concentrations correlated particularly with frontal lobe atrophy rate (r = 0.53, p = 0.003). Conclusions: Increased serum NfL concentrations are seen in FTD but show wide variability within each clinical and genetic group. Higher concentrations may reflect the intensity of the disease in FTD and are associated with more rapid atrophy of the frontal lobes. PMID:27581216

  19. [Immunoglobulin genes in lymphoid cells and regulation of their transcription].

    PubMed

    Stepchenko, A G; Urakov, D N; Luchina, N N; Deev, S M; Polianovskiĭ, O L

    1990-01-01

    The hybridoma genomes contain polyploid sets of immunoglobulin genes. We have shown, that the hybridoma PTF-02 genome contains three genes of heavy chains and two genes of light chains. The genes responsible for antibody synthesis were cloned and their structure were determined. Investigation of the kappa gene transcription and its fragments which contain regulatory sequences revealed a nuclear factor. The latter interacts with the octanucleotide localized at the promoter region of the kappa gene. The purified factor activates the transcription of the kappa gene in a heterologous cell-free system. Together with the tissue-specific factor there is also an universal factor interacting with the octanucleotide sequence. We have shown an additional factor in lymphoid cells interact with the protein which binds to the octanucleotide sequence. We have shown an additional factor in lymphoid cells interacting with the protein which binds to the octanucleotide sequence. As a result, there is a family of factors which interact with ATTTGCAT sequence. One major factor (m.w. 60 +/- 2 kDa) is an obligatory component for the initiation of immunoglobulin genes transcription.

  20. Membrane IgD-positive B cells of "low-IgD serum phenotype" individuals fail to secrete IgD and fail to shift to preferential lambda light-chain expression in vitro.

    PubMed

    Litwin, S D; Zehr, B D

    1987-03-01

    IgD production by short-term human peripheral blood mononuclear cell (PBM) cultures was studied to establish the in vitro correlates of low serum IgD expression. Cells of persons with less than 3 micrograms/ml IgD in the serum, referred to as the low-serum IgD phenotype (LISP), were analyzed. Advantage was taken of recently developed data on spontaneous IgD biosynthesis by human B cells and the observation that lambda light chains are preferentially expressed by IgD-secreting cells in vitro. Initial analysis of an IgD serum distribution showed that all LISP sera contained low but detectable amounts of IgD, with a mean value of 0.85 microgram/ml; this figure was 30- to 35-fold lower than the mean of the majority of the population. LISP PBM contained normal numbers of IgD-positive B cells which displayed a normal intensity of IgD per cell using comparative analysis of mean channel fluorescence by cell flow cytometry. Several lines of evidence suggested that IgD-secreting cells could not be generated from LISP lymphocytes in vitro. Namely, it was found that no IgD immunoglobulin-containing cells were found among PBM of LISP persons; cell lysates enriched for the intracellular fraction by Triton X-114 phase separation showed low IgD in LISP cells despite "normal" amounts of IgD in membrane-enriched fraction preparations; there was no spontaneous IgD secretion by any LISP PBM cultures; and neither LISP sera nor cellular IgD preparations showed IgD lambda/kappa ratios greater than 1.0, indicative of the absence of the preferential lambda light-chain expression associated with secretion of IgD.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3106400

  1. [Seasonal changes in phosphorylation of myosin regulatory light chains and C-protein in myocardium of hibernating ground squirrel Citellus undulatus].

    PubMed

    Malyshev, S L; Osipova, D A; Vikhliantsev, I M; Podlubnaia, Z A

    2006-01-01

    A comparative study concerning the extent of phosphorylation of myosin regulatory light chains and C-protein from the left ventricle of hibernating ground squirrel Citellus undulatus during the periods of hibernation and activity was carried out. During hibernation, regulatory light chains of ground squirrel were found to be completely dephosphorylated. In active animals, the share of phosphorylated light chains averages 40-45% of their total amount. The extent of phosphorylation of the cardiac C-protein during hibernation is about two times higher than that in the active state. Seasonal differences in phosphorylation of the two proteins of ground squirrel myocardium are discussed in the context of adaptation to hibernation.

  2. Identification of secreted and membrane-bound bat immunoglobulin using a Microchiropteran-specific mouse monoclonal antibody.

    PubMed

    Lee, William T; Jones, Derek D; Yates, Jennifer L; Winslow, Gary M; Davis, April D; Rudd, Robert J; Barron, Christopher T; Cowan, Cailyn

    2016-12-01

    Bat immunity has received increasing attention because some bat species are being decimated by the fungal disease, White Nose Syndrome, while other species are potential reservoirs of zoonotic viruses. Identifying specific immune processes requires new specific tools and reagents. In this study, we describe a new mouse monoclonal antibody (mAb) reactive with Eptesicus fuscus immunoglobulins. The epitope recognized by mAb BT1-4F10 was localized to immunoglobulin light (lambda) chains; hence, the mAb recognized serum immunoglobulins and B lymphocytes. The BT1-4F10 epitope appeared to be restricted to Microchiropteran immunoglobulins and absent from Megachiropteran immunoglobulins. Analyses of sera and other E. fuscus fluids showed that most, if not all, secreted immunoglobulins utilized lambda light chains. Finally, mAb BT1-4F10 permitted the identification of B cell follicles in splenic white pulp. This Microchiropteran-specific mAb has potential utility in seroassays; hence, this reagent may have both basic and practical applications for studying immune process. PMID:27377583

  3. Omega-3 and dyslexia

    PubMed Central

    Zelcer, Michal; Goldman, Ran D.

    2015-01-01

    Abstract Question In light of the increase in the number of school-aged children diagnosed with dyslexia, what is the role of omega-3 supplements in the management of this condition? Answer Dyslexia is the most common learning disability and is known to have multifactorial causes. Recent evidence suggests that there is a connection between defects in highly unsaturated fatty acid metabolism and neurodevelopmental disorders such as dyslexia. While the benefit of omega-3 supplementation for children with dyslexia has been studied, evidence remains limited. Unified diagnostic criteria for dyslexia, objective measures of fatty acid deficiency, and close monitoring of dietary intake are some of the factors that would improve the quality of research in the field. PMID:26371100

  4. Roles of heavy and light chains in IgM polymerization.

    PubMed Central

    Bornemann, K D; Brewer, J W; Beck-Engeser, G B; Corley, R B; Haas, I G; Jäck, H M

    1995-01-01

    IgM antibodies are secreted as multisubunit polymers that consist of as many as three discrete polypeptides: mu heavy chains, light (L) chains, and joining (J) chains. We wished to determine whether L chains that are required to confer secretory competence on immunoglobulin molecules must be present for IgM to polymerize--that is, for intersubunit disulfide bonds to form between mu chains. Using a L-chain-loss variant of an IgM-secreting hybridoma, we demonstrated that mu chains were efficiently polymerized independent of L chains, in a manner similar to that observed for conventional microL complexes, and that the mu polymers incorporated J chain. These mu polymers were not secreted but remained associated with the endoplasmic reticulum-resident chaperone BiP (GRP78). This finding is consistent with the endoplasmic reticulum being the subcellular site of IgM polymerization. We conclude that mu chain alone has the potential to direct the polymerization of secreted IgM, a process necessary but not sufficient for IgM to attain secretory competence. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7761423

  5. Biology of Immunoglobulins

    PubMed Central

    Berlot, Giorgio; Rossini, Perla; Turchet, Federica

    2015-01-01

    Intravenous Immunoglobulins (IvIg) are often administered to critically ill patients more as an act of faith than on the basis of relevant clinical studies. This particularly applies to the treatment of sepsis in adult patients, in whom the current guidelines even recommend against their use, despite that many studies demonstrated either their beneficial effects in different subsets of patients and that some preparations of IvIg are more effective than other. The biology of Ig are reviewed, aiming to a more in-depth understanding of their properties in order to clarify their possible indications in different clinical settings. PMID:25674545

  6. The importance of screening for serum free light chains in suspected cases of multiple myeloma and their impact on the kidney

    PubMed Central

    Talbot, B; Wright, D; Basnayake, K

    2014-01-01

    Multiple myeloma (MM) is the second most common haematological malignancy in the UK. We present a case series of three patients with light chain only myeloma who had normal serum protein electrophoretic studies at screening and were diagnosed using serum and urine free light chain assessment. This series reiterates the importance of thorough and robust screening for MM in patients presenting with renal disease. We review the up to date literature and we highlight the need to screen patients for MM with a combination of serum electrophoresis/immunofixation and either urinary or serum free light chain measurement and to maintain a high index of suspicion regardless of the presence or absence of proteinuria. We also discuss the emerging role of the serum free light chain assay. PMID:25326567

  7. Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

    PubMed

    Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

    2015-07-01

    Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab.

  8. Myosin light chain phosphorylation in sup 32 P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine

    SciTech Connect

    Singer, H.A.; Oren, J.W.; Benscoter, H.A. )

    1989-12-15

    The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.

  9. Quantitative analysis of the free energy coupling in the system calmodulin, calcium, smooth muscle myosin light chain kinase.

    PubMed

    Mamar-Bachi, A; Cox, J A

    1987-12-01

    Interactions between Ca2+, calmodulin and turkey gizzard myosin light chain kinase have been studied by equilibrium gel filtration and analyzed in terms of the theory of free energy coupling as formulated by Huang and King for calmodulin-regulated systems (Current Topics in Cellular Regulation 27, 1966-1971, 1985). Direct binding studies revealed that upon interaction with the enzyme, calmodulin acquires strong positive cooperativity in Ca2+-binding. The determination of the Ca2+-binding constants is inherently approximative due to the apparent homotropic cooperativity; therefore a statistical chi 2 analysis was carried out to delimit the formation-, and subsequently the stoichiometric Ca2+-binding constants. Whereas the first two stoichiometric Ca2+-binding constants of enzyme-bound CaM do not differ or are at the upmost 10-fold higher than those in free calmodulin, the third Ca2+ ion binds with an at least 70-fold and more likely 3000-fold higher affinity constant. The binding constant for the fourth Ca2+ is only 5-fold higher than the corresponding one in free calmodulin, thus creating a plateau at 3 bound Ca2+ in the isotherm. Direct binding of Ca2+-free calmodulin to myosin light chain kinase at 10(-7) M free Ca2+ yielded a l/l stoichiometry and an affinity constant of 2.2 x 10(5) M-1. It is thus anticipated that in resting smooth muscle ([Ca2+] less than or equal to 10(-7) M) more than half of the enzyme is bound to metal-free calmodulin. Analysis of the enzymatic activation of myosin light chain kinase at different concentrations of calmodulin and Ca2+ revealed that this Ca2+-free complex is inactive and that activation is concomitant with the formation of the enzyme.calmodulin.Ca3 complex.

  10. [Effect of myosin alkali light chains on myosin subfragment 1 interaction with actin in solution and in ghost muscle fiber].

    PubMed

    Levistkiĭ, D I; Borovikov, Iu S; Nikolaeva, O P; Golitsyna, N L; Poglazov, B F

    1990-09-01

    At low ionic strength (7-25 mM) Mg2(+)-ATPase of myosin subfragment 1 (S1) isoforms containing alkali light chain A1 [S1(A1)] is activated by actin 1.5-2.5 times as strongly as Mg2(+)-ATPase of S1 isoforms containing alkali light chain A2[S1(A2)]. Data from analytical ultracentrifugation suggest that at low ionic strength in the absence of ATP in solution S1(A1) displays a higher affinity for F-actin than S1(A2). Such a higher affinity of S1(A1) for F-actin was also demonstrated by experiments, in which the interaction of S1 isoforms fluorescently labeled by 1.5-IAEDANS with F-actin of ghost fibers (single glycerinated muscle fibers containing F-actin but devoid of myosin) was studied. Using polarization microfluorimetry, it was shown that the interaction of both S1 isoforms with ghost fiber F-actin induces similar changes in the parameters of polarized tryptophan fluorescence. At the same time the mobility of the fluorescent probe, 1.5-IAEDANS, specifically attached to the SH-group of Cys-374 in the C-terminal region of action is markedly decreased by S1(A1) and is only slightly affected by S1(A2). The data obtained suggest that S1(A1) and S1(A2) interact with the C-terminal region of the actin molecule in different ways, i.e. S1(A1) is attached more firmly than S1(A2). This may be due to the existence of contacts between the alkali light chain of A1 of S1(A1) and the C-terminal region of actin as well as to the absence of such contacts in the case of S1(A2).

  11. β-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration

    PubMed Central

    Simard, Elie; Kovacs, Jeffrey J.; Miller, William E.; Kim, Jihee; Grandbois, Michel; Lefkowitz, Robert J.

    2013-01-01

    Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the β-arrestin biased ligand [Sar1,Ile4,Ile8]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility. PMID:24255721

  12. Immunoglobulin allotypes and immunoglobulin G subclass responses to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in early-onset periodontitis.

    PubMed Central

    Choi, J I; Ha, M H; Kim, J H; Kim, S J

    1996-01-01

    The present study was performed to estimate the observed frequencies of the immunoglobulin heavy-chain (Gm) and light-chain (Km) allotypes among patients with early-onset periodontitis (EOP) and their effect on the IgG2 subclass responses against Actinobacillus actinomycetemcomitans Y4 and Porphyromonas gingivalis 381, respectively. Sixty-nine EOP patients, including 11 with localized juvenile periodontitis (LJP), 19 who had LJP, 15 with LJP-rapidly progressing periodontitis (RPP), and 24 with RPP, were examined for the Gm and Km allotypes by a hemagglutination inhibition test. Levels of immunoglobulin G2 (IgG2) antibodies against the two organisms were determined by enzyme-linked immunosorbent assay. Fifty race- and age-matched, periodontally healthy subjects were also included as a control group. The observed frequencies of the Gm haplotype afnb and Km(1) were significantly higher in the RPP and LJP groups, respectively. The G2m(n)+ group of those with RPP and the Km(1)+ group of those with LJP had significantly higher levels of IgG2 antibodies to A. actinomycetemcomitans and P. gingivalis, respectively. The results indicate that linkage disequilibrium of the G2m(n) locus in RPP patients or the Km(1) locus in LJP patients may be associated with high IgG2 antibody responses to the respective bacteria. It was reasoned that the IgG2 antibody responses are associated with the immunoglobulin allotypes. The function of IgG2 antibodies in their reaction to different bacterial antigens may be interpreted as either protective or nonprotective in the two different types of EOP (i.e., LJP and RPP). PMID:8926092

  13. Damper Spring For Omega Seal

    NASA Technical Reports Server (NTRS)

    Maclaughlin, Scott T.; Montgomery, Stuart K.

    1993-01-01

    Damper spring reduces deflections of omega-cross-section seal, reducing probability of failure and extending life of seal. Spring is split ring with U-shaped cross section. Placed inside omega seal and inserted with seal into seal cavity. As omega seal compressed into cavity, spring and seal make contact near convolution of seal, and spring becomes compressed also. During operation, when seal dynamically loaded, spring limits deflection of seal, reducing stress on seal.

  14. Renal Light Chain Deposition Associated with the Formation of Intracellular Crystalline Inclusion Bodies in Podocytes: A Rare Case Report.

    PubMed

    Wang, Yuan-da; Dong, Zhe-yi; Zhang, Xue-guang; Zhang, Wei; Yin, Zhong; Qiu, Qiang; Chen, Xiang-mei

    2016-01-01

    We herein report the case of an elderly woman with bone pain and proteinuria as the main clinical manifestations. The patient was diagnosed with the IgG κ type of multiple myeloma. Her renal pathology consisted of widespread κ light chain protein deposition associated with the formation of large quantities of rod-like crystals in podocytes. This phenomenon is very rare. We explored the significance of this crystal formation via a detailed and descriptive analysis and also performed a literature review, thus providing data to increase the available information about this type of disease.

  15. HED physics frontiers on OMEGA/OMEGA EP

    NASA Astrophysics Data System (ADS)

    Meyerhofer, D. D.

    2008-04-01

    The 60 beam, 30 kJ, OMEGA laser facility has been operating at the University of Rochester for more than a decade. The OMEGA EP laser facility adjacent to it will be completed in Q3FY08. OMEGA EP will consist of four beamlines with NIF-like architecture. Each of the beams will ultimately produce 10 ns 6.5 kJ energy ultraviolet pulses directed into the EP target chamber. Two of the beamlines will also operate as high energy petawatt (HEPW) lasers, with up to 2.6 kJ each in 10 ps IR pulses. The HEPW beams can be injected into either the EP chamber or the existing OMEGA target chamber for integrated experiments. This talk will describe the OMEGA EP project status, HED physics possibilities using the combined system, and opportunities for external user access. The full OMEGA laser system (original 60 beam OMEGA and OMEGA EP) will allow unprecedented opportunities for HED physics research. These include backlighting of ICF implosions and integrated Fast Ignition Experiments in the OMEGA target chamber. The configuration flexibility of the OMEGA EP target area, will allow a wide variety of HED physics research, with the possibility of mixing and matching short and long pulse laser beams. Research areas will include episodic jets, the use of up to 40 ns long drive pulses for Equation of State measurements, short pulse, high intensity backlighting of otherwise opaque materials, and the possibility of creating an electron-positron plasma The ongoing OMEGA EP Use Planning process will be described.

  16. Immunoglobulin profile in schizophrenia.

    PubMed

    Bhatia, M S; Dhar, N K; Agrawal, P; Khurana, S K; Neena, B; Malik, S C

    1992-08-01

    The present study was conducted on 40 new consecutive schizophrenic patients admitted in the psychiatry ward. The diagnosis of schizophrenia was done by Research Diagnosis Criteria (RDC). Serum immunoglobulins were were estimated in schizophrenic patients and were age and sex matched with 40 healthy individuals, comprising the control group. The IgG and IgA mean levels of schizophrenic patients were found to be significantly higher (p < 0.01) than the normal healthy individuals. There were however no significant differences between the schizophrenic patients and control group regarding total proteins, albumin and globulin levels. In subtypes of schinophrenia based on phenomenology only, paranoid group scored significantly higher (p < 0.01) IgG and IgA mean values than other types of Schizophrenia (catatonic, disorganised and undifferentiated).

  17. TNF causes changes in glomerular endothelial permeability and morphology through a Rho and myosin light chain kinase-dependent mechanism.

    PubMed

    Xu, Chang; Wu, Xiaoyan; Hack, Bradley K; Bao, Lihua; Cunningham, Patrick N

    2015-12-01

    A key function of the endothelium is to serve as a regulated barrier between tissue compartments. We have previously shown that tumor necrosis factor (TNF) plays a crucial role in lipopolysaccharide (LPS)-induced acute kidney injury, in part by causing injury to the renal endothelium through its receptor TNFR1. Here, we report that TNF increased permeability to albumin in primary culture mouse renal endothelial cells, as well as human glomerular endothelial cells. This process occurred in association with changes in the actin cytoskeleton and was associated with gaps between previously confluent cells in culture and decreases in the tight junction protein occludin. This process was dependent on myosin light chain activation, as seen by its prevention with Rho-associated kinase and myosin light chain kinase (MLCK) inhibitors. Surprisingly, permeability was not blocked by inhibition of apoptosis with caspase inhibitors. Additionally, we found that the renal glycocalyx, which plays an important role in barrier function, was also degraded by TNF in a Rho and MLCK dependent fashion. TNF treatment caused a decrease in the size of endothelial fenestrae, dependent on Rho and MLCK, although the relevance of this to changes in permeability is uncertain. In summary, TNF-induced barrier dysfunction in renal endothelial cells is crucially dependent upon the Rho/MLCK signaling pathway.

  18. Reconstitution of heavy chain and light chain 1 in cardiac subfragment-1 from hyperthyroid and euthyroid rabbit hearts.

    PubMed

    Ueda, S; Yamaoki, K; Nagai, R; Yazaki, Y

    1983-01-01

    It is now established that cardiac myosin from hyperthyroid rabbit hearts (TXM) exhibits high Ca2+ ATPase activity. The high Ca2+ ATPase activity of TXM was completely retained in cardiac myosin subfragment-1 (S-1) (1.33 +/- 0.04 mumol Pi/mg per min; euthyroid, 0.51 +/- 0.04). Cardiac S-1 from hyperthyroid and euthyroid rabbits (TXS-1 and NS-1) had the same pattern in SDS-polyacrylamide gel electrophoresis. The possible influence of heavy and light chains of TXM on increasing the ATPase activity was examined by reconstitution in the S-1 preparation. Crosswise reconstitution was performed using cardiac S-1 heavy chain (90,000 daltons) and light chain 1 (LC1) (27,000 daltons) from hyperthyroid and euthyroid hearts. Reconstitution was verified by using radiolabeled LC1. More than 95% of S-1 was recovered with full ATPase activity. When TXS-1 was reconstituted with LC1 from euthyroid hearts, the reconstituted molecule retained high ATPase activity. On the other hand, NS-1 reconstituted with LC1 from hyperthyroid hearts failed to increase the ATPase activity. The ATPase activity of S-1 was determined by the source of the heavy chain. These results suggest that the high Ca2+ ATPase activity of cardiac myosin and S-1 from hyperthyroid animals arises from the molecular alteration of the heavy chain induced by thyroxine administration. PMID:6304826

  19. IgD multiple myeloma: Clinical, biological features and prognostic value of the serum free light chain assay.

    PubMed

    Djidjik, R; Lounici, Y; Chergeulaïne, K; Berkouk, Y; Mouhoub, S; Chaib, S; Belhani, M; Ghaffor, M

    2015-09-01

    IgD multiple myeloma (MM) is a rare subtype of myeloma, it affects less than 2% of patients with MM. To evaluate the clinical and prognostic attributes of serum free light chains (sFLCs) analysis, we examined 17 cases of IgD MM. From 1998 to 2012, we obtained 1250 monoclonal gammapathies including 590 multiple myeloma and 17 patients had IgD MM. With preponderance of men patients with a mean age at diagnosis of: 59±12years. Patients with IgD MM have a short survival (Median survival=9months). The presenting features included: bone pain (75%), lymphadenopathy (16%), hepatomegaly (25%), splenomegaly (8%), associated AL amyloidosis (6%), renal impairment function (82%), infections (47%), hypercalcemia (37%) and anemia (93%). Serum electrophoresis showed a subtle M-spike (Mean=13.22±10g/L) in all patients associated to a hypogammaglobulinemia. There was an over-representation of Lambda light chain (65%); high serum β2-microglobulin in 91% and Bence Jones proteinuria was identified in 71%. The median rate of sFLCs κ was 19.05mg/L and 296.75mg/L for sFLCs λ. sFLCR was abnormal in 93% of patients and it showed concordance between baseline sFLCR and the survival (P=0.034). The contribution of FLC assay is crucial for the prognosis of patients with IgD MM.

  20. European trial of free light chain removal by extended haemodialysis in cast nephropathy (EuLITE): A randomised control trial

    PubMed Central

    Hutchison, Colin A; Cook, Mark; Heyne, Nils; Weisel, Katja; Billingham, Lucinda; Bradwell, Arthur; Cockwell, Paul

    2008-01-01

    Background Renal failure is a frequent complication of multiple myeloma and when severe is associated with a greatly increased morbidity and mortality. The principal cause of severe renal failure is cast nephropathy, a direct consequence of high concentrations of monoclonal free light chains (FLCs) in patients' sera. FLC removal by extended haemodialysis, using a high cut-off dialyser, has recently been described as a novel therapeutic option. Methods The EUropean trial of free LIght chain removal by exTEnded haemodialysis in cast nephropathy (EuLITE) trial is a prospective, randomised, multicentre, open label clinical trial to investigate the clinical benefits of FLC removal haemodialysis in patients with cast nephropathy, dialysis dependent acute renal failure and de novo multiple myeloma. Recruitment commenced in May 2008. In total, 90 patients will be recruited. Participants will be randomised, centrally, upon enrolment, to either trial chemotherapy and FLC removal haemodialysis or trial chemotherapy and standard high flux haemodialysis. Trial chemotherapy consists of bortezomib, doxorubicin and dexamethasone. FLC removal haemodialysis is undertaken with two Gambro HCO 1100 dialysers in series using an intensive treatment schedule. The primary outcome for the study is independence of dialysis at 3 months. Secondary outcomes are: duration of dialysis, reduction in serum FLC concentrations; myeloma response and survival. Hypothesis FLC removal haemodialysis will increase the rate of renal recovery in patients with severe renal failure secondary to cast nephropathy in de novo multiple myeloma. Trial registration ISRCTN45967602 PMID:18822172

  1. The importance of complete tissue homogenization for accurate stoichiometric measurement of myosin light chain phosphorylation in airway smooth muscle.

    PubMed

    Wang, Lu; Paré, Peter D; Seow, Chun Y

    2015-02-01

    The standard method for measuring the phosphorylation of the regulatory myosin light chain (MLC20) in smooth muscle is extraction of the light chain using a urea extraction buffer, urea-glycerol gel electrophoresis of the soluble portion of the extract (supernatant) and Western blot analysis. The undissolved portion of the tissue during extraction (the pellet) is usually discarded. Because the pellet contains a finite amount of MLC20, omission of the pellet could result in inaccurate measurement of MLC20 phosphorylation. In this study we compared the level of tracheal smooth muscle MLC20 phosphorylation in the supernatant alone, with that in the complete tissue homogenate (supernatant and pellet) using the standard method. The supernatant fraction showed the well-known double bands representing phosphorylated and un-phosphorylated MLC20. The dissolved pellet fraction showed varying amounts of un-phosphorylated and phosphorylated MLC20. There was a small but statistically significant overestimation of the percent MLC20 phosphorylation if the pellet was not taken into consideration. The overestimation was 7% ± 2% (mean ± SEM) (p < 0.05) in unstimulated muscle and 2% ± 1% (p < 0.05) in acetylcholine (10(-6) mol/L) stimulated muscle. This finding suggests that for accurate estimation of the stoichiometry of MLC20 phosphorylation it is necessary to consider the contribution from the pellet portion of the muscle tissue homogenate.

  2. [The concurrence of light-chain deposition disease, AL-amyloidosis, and cast nephropathy in a patient with multiple myeloma].

    PubMed

    Rekhtina, I G; Zakharova, E V; Stoliarevich, E S; Sinitsina, M N; Denisova, E N

    2015-01-01

    Despite of the fact that their clinical manifestations are similar, AL-amyloidosis (AL-A) and light chain deposition disease (LCDD) are individual nosological entities in view of considerable differences in their pathogenesis and pathomorphology. The paper describes a rare case of the concurrence of LCDD and AL-A in a patient with multiple myeloma. Clinically, there was dialysis-dependent renal failure, flail leg syndrome, myocardiopathy, and rhabdomyolysis. At the disease onset, his nephrobiopsy specimen could diagnose LCDD and myeloma or cast nephropathy. The disease was characterized by an aggressive course. Despite the administration of innovative agents, the patient had a short-term remission and died from disease progression. Autopsy additionally revealed amyloid deposition in the heart and kidney. The development of AL-A in the presence of prior LCDD may reflect the progression of the tumor and the appearance of an additional subclone of plasma cells that produce amyloidogenic light chains. The uncommonness of this case is that renal amyloid was found in the tubular casts and absent in the glomeruli, which may be considered as a special form--tubular AL-amyloidosis. PMID:26281203

  3. Surrogate light chain expression beyond the pre-B cell stage promotes tolerance in a dose-dependent fashion.

    PubMed

    Kil, Laurens P; Corneth, Odilia B J; de Bruijn, Marjolein J W; Asmawidjaja, Patrick S; Krause, Arndt; Lubberts, Erik; van Loo, Pieter Fokko; Hendriks, Rudi W

    2015-02-01

    While surrogate light chain (SLC) expression is normally terminated in differentiating pre-B cells, co-expression of SLC and conventional light chains has been reported in a small population of autoreactive peripheral human B cells that accumulate in arthritic joints. Despite this association with autoimmunity the contribution of SLC expressing mature B cells to disease development is still unknown. We studied the pathogenicity of SLC(+) B cells in a panel of mice that transgenically express the SLC components VpreB and λ5 throughout B cell development. Here we report that although VpreB or λ5 expression mildly activated mature B cells, only moderate VpreB expression levels - in the absence of λ5 - enhanced IgG plasma cell formation. However, no autoantibody production was detectable in VpreB or λ5 transgenic mice and VpreB expression could not accelerate autoimmunity. Instead, moderate VpreB expression partially protected mice from induced autoimmune arthritis. In support of a tolerogenic role of SLC-transgenic B cells, we observed that in a dose-dependent manner SLC expression beyond the pre-B cell stage enhanced clonal deletion among immature and transitional B cells and rendered mature B cells anergic. These findings suggest that SLC expression does not propagate autoimmunity, but instead may impose tolerance.

  4. TNF causes changes in glomerular endothelial permeability and morphology through a Rho and myosin light chain kinase-dependent mechanism.

    PubMed

    Xu, Chang; Wu, Xiaoyan; Hack, Bradley K; Bao, Lihua; Cunningham, Patrick N

    2015-12-01

    A key function of the endothelium is to serve as a regulated barrier between tissue compartments. We have previously shown that tumor necrosis factor (TNF) plays a crucial role in lipopolysaccharide (LPS)-induced acute kidney injury, in part by causing injury to the renal endothelium through its receptor TNFR1. Here, we report that TNF increased permeability to albumin in primary culture mouse renal endothelial cells, as well as human glomerular endothelial cells. This process occurred in association with changes in the actin cytoskeleton and was associated with gaps between previously confluent cells in culture and decreases in the tight junction protein occludin. This process was dependent on myosin light chain activation, as seen by its prevention with Rho-associated kinase and myosin light chain kinase (MLCK) inhibitors. Surprisingly, permeability was not blocked by inhibition of apoptosis with caspase inhibitors. Additionally, we found that the renal glycocalyx, which plays an important role in barrier function, was also degraded by TNF in a Rho and MLCK dependent fashion. TNF treatment caused a decrease in the size of endothelial fenestrae, dependent on Rho and MLCK, although the relevance of this to changes in permeability is uncertain. In summary, TNF-induced barrier dysfunction in renal endothelial cells is crucially dependent upon the Rho/MLCK signaling pathway. PMID:26634902

  5. Crystal structure of human dynein light chain Dnlc2A: Structural insights into the interaction with IC74

    SciTech Connect

    Liu Junfeng; Wang Zhanxin; Wang Xinquan; Tang Qun; An Xiaomin; Gui Lulu; Liang Dongcai . E-mail: dcliang@sun5.ibp.ac.cn

    2006-10-27

    The human light chain of the motor protein dynein, Dnlc2A, is also a novel TGF-{beta}-signaling component, which is altered with high frequency in epithelial ovarian cancer. It is an important mediator of dynein and the development of cancer, owing to its ability to bind to the dynein intermediate light chain (DIC) IC74 and to regulate TGF-{beta}-dependent transcriptional events. Here we report the 2.1-A crystal structure of Dnlc2A using single anomalous diffraction. The proteins form a homodimer in solution and interact mainly through the helix {alpha}{sub 2}, strand {beta}{sub 3}, and the loop following this strand in each protein to generate a 10-stranded {beta}-sheet core. The surface of the {beta}-sheet core is mainly positively charged and predicted (by software PPI-Pred) to be the site that interacts with other partners. At the same time, the residues 79-82, 88, and 90 of each molecule formed two holes in the core. Residue 89 of each molecule, which is crucial for the DIC binding function of Dnlc2A, is within the holes. On the basis of these observations, we propose that the homodimer is the structural and functional unit maintained by hydrogen bonding interactions and hydrophobic packing, and that the patch of the surface of the {beta}-sheet core is the main area of interaction with other partners. Furthermore, the two holes would be the key sites to interact with IC74.

  6. Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes.

    PubMed

    Varma, Dileep; Dawn, Amrita; Ghosh-Roy, Anindya; Weil, Sarah J; Ori-McKenney, Kassandra M; Zhao, Yanqiu; Keen, James; Vallee, Richard B; Williams, John C

    2010-02-23

    The ability to rapidly and specifically regulate protein activity combined with in vivo functional assays and/or imaging can provide unique insight into underlying molecular processes. Here we describe the application of chemically induced dimerization of FKBP to create nearly instantaneous high-affinity bivalent ligands capable of sequestering cellular targets from their endogenous partners. We demonstrate the specificity and efficacy of these inducible, dimeric "traps" for the dynein light chains LC8 (Dynll1) and TcTex1 (Dynlt1). Both light chains can simultaneously bind at adjacent sites of dynein intermediate chain at the base of the dynein motor complex, yet their specific function with respect to the dynein motor or other interacting proteins has been difficult to dissect. Using these traps in cultured mammalian cells, we observed that induction of dimerization of either the LC8 or TcTex1 trap rapidly disrupted early endosomal and lysosomal organization. Dimerization of either trap also disrupted Golgi organization, but at a substantially slower rate. Using either trap, the time course for disruption of each organelle was similar, suggesting a common regulatory mechanism. However, despite the essential role of dynein in cell division, neither trap had a discernable effect on mitotic progression. Taken together, these studies suggest that LC occupancy of the dynein motor complex directly affects some, but not all, dynein-mediated processes. Although the described traps offer a method for rapid inhibition of dynein function, the design principle can be extended to other molecular complexes for in vivo studies.

  7. Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach.

    PubMed

    Chang, Tzuu-Wang; Janardhanan, Pavithra; Mello, Charlene M; Singh, Bal Ram; Cai, Shuowei

    2016-09-01

    Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, a non-radioactive-based systematic evolution of ligands by exponential enrichment (SELEX) process is developed by utilizing surface plasmon resonance to monitor the binding enrichment. Two RNA aptamers have been identified as strong binders against light chain of botulinum neurotoxin type A. These two aptamers showed strong inhibition activity on LCA, with IC50 in nanomolar range. Inhibition kinetic studies reveal mid nanomolar KI and non-competitive nature of their inhibition, suggesting that they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we observed that the 2'-fluorine-pyrimidine-modified RNA aptamers identified here do not change their binding and biological activities. This observation could lead to a cost-effective way for SELEX, by using regular nucleotide during SELEX, and 2'-fluorine-pyrimidine-modified nucleotide for final application to enhance their RNase-resistance. PMID:27085355

  8. Polar drive on OMEGA

    NASA Astrophysics Data System (ADS)

    Radha, P. B.; Marshall, F. J.; Boehly, T. R.; Collins, T. J. B.; Craxton, R. S.; Edgell, D.; Epstein, R.; Frenje, J.; Goncharov, V. N.; Marozas, J. A.; McCrory, R. L.; McKenty, P. W.; Meyerhofer, D. D.; Petrasso, R. D.; Sangster, T. C.; Shvydky, A.; Skupsky, S.

    2013-11-01

    High-convergence polar-drive experiments are being conducted on OMEGA [T. R. Boehly et al., Opt. Commum. 133, 495 (1997)] using triple-picket laser pulses. The goal of OMEGA experiments is to validate modeling of oblique laser deposition, heat conduction in the presence of nonradial thermal gradients in the corona, and implosion energetics in the presence of laser-plasma interactions such as crossed-beam energy transfer. Simulated shock velocities near the equator, where the beams are obliquely incident, are within 5% of experimentally inferred values in warm plastic shells, well within the required accuracy for ignition. High, near-one-dimensional areal density is obtained in warm-plastic-shell implosions. Simulated backlit images of the compressing core are in good agreement with measured images. Outstanding questions that will be addressed in the future relate to the role of cross-beam transfer in polar drive irradiation and increasing the energy coupled into the target by decreasing beam obliquity.

  9. Immunoglobulin Resistance in Kawasaki Disease

    PubMed Central

    Hartas, Georgios A.; Hashmi, Syed Shahrukh; Pham-Peyton, Chi; Tsounias, Emmanouil; Bricker, John T.

    2015-01-01

    Background: The aim of this study was to identify risk factors for immunoglobulin resistance, including clinical symptoms such as arthritis and the pH of intravenous immunoglobulin. Methods: The data of children with Kawasaki disease who had received immunoglobulin were evaluated. Data regarding the brand of immunoglobulin administered were abstracted from the pharmacy records. Results: Eighty consecutive children with Kawasaki disease were evaluated (Mdnage=28 months, 66% male). The prevalence of immunoglobulin resistance was 30%. Arthritis was a presenting symptom in the acute phase of Kawasaki disease in 8% (6/80, all male) and was seen in significant association with immunoglobulin resistance in comparison to those without arthritis (16.7% vs. 0.2%, p=0.008). Next, the immunoglobulin brand types were divided into two groups: the relatively high pH group (n=16), including Carimune (pH 6.6±0.2), and the low pH group (n=63), including Gamunex (pH 4–4.5) or Privigen (pH 4.6–5). Overall, no significant difference in immunoglobulin responsiveness was found between the low pH and the high pH groups (73% vs. 56%, p=0.193), although the low pH group showed a trend toward a larger decrease in erythrocyte sedimentation rate (p=0.048), lower steroid use (p=0.054), and lower coronary involvement (p=0.08) than those in the high pH group. Conclusions: Children presenting with arthritis in the acute phase of Kawasaki disease may be at risk for immunoglobulin resistance. PMID:25852966

  10. C57BL/6 x BALB/c hybridomas produce IgA which assembles into molecules with covalent bonds between heavy chains (H) and light chains (L), and into molecules lacking covalent bonds between H and L.

    PubMed

    Wims, L A; Sharon, J; Newman, B; Kabat, E A; Morrison, S L

    1985-12-01

    Examination of the gel electrophoresis patterns of 14C-biosynthetically labeled immunoglobulin from C57BL/6 X BALB/c IgA hybridomas reveals that each of the monoclonal cell populations produces two different forms of IgA: molecules with heavy chains (H) and light chains (L) joined by disulfide bonds, as well as molecules with H and L being noncovalently associated. The possible origin of this was explored: Southern blot analysis of the hybridoma DNA indicated that only one alpha gene is expressed by each cell line; hybridoma cells labeled in the presence of the N-glycosylation inhibitor tunicamycin exhibit both forms; and electrophoresis of biosynthetically labeled spleen cell IgA from C57BL/6, BALB/c and (C57BL/6 X BALB/c) F1 mice shows that BALB/c mice produce only the noncovalently associated form, while C57BL/6 and (C57BL/6 X BALB/c) F1 mice produce both. Possible mechanisms by which two types of IgA may be assembled by the same hybridoma cell are discussed.

  11. Beta-2-glycoprotein specificity of human anti-phospholipid antibody resides on the light chain: a novel mechanism for acquisition of cross-reactivity by an autoantibody.

    PubMed

    Kumar, Sanjeev; Nagl, Sylvia; Kalsi, Jatinderpal K; Ravirajan, Chelliah T; Athwal, Dee; Latchman, David S; Pearl, Laurence H; Isenberg, David A

    2005-01-01

    We have recently shown that the anti-cardiolipin activity of human anti-phospholipid antibody UK4 (lambda) resides on its heavy chain. We now show that UK4 possesses strong reactivity to the plasma-protein beta2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 (kappa) with no reactivity to beta2-GPI, we now demonstrate that both the constructs possessing the auto-antibody-derived light chain exhibited significant binding to beta2-GPI. However, the construct possessing UK4 heavy chain in association with 4D5 light chain, exhibited no anti-beta2-GPI activity. Furthermore, there was a low increase (approximately 10%) in the binding of UK4 to cardiolipin in the presence of beta2-GPI. The results demonstrate that anti-beta2-GPI activity resides on UK4 light chain and, importantly, this activity could be transferred to a novel antibody construct via the light chain alone. Computer-generated models of the three-dimensional structures of UK4 and its hybrids, suggest predominant interaction of UK4 light chain with domain IV of beta2-GPI. Molecular docking experiments highlight a number of potential sites on beta2-GPI for interaction of UK4 and indicate as to how beta2-GPI recognition may occur primarily via the autoantibody light chain. The study provides first demonstration of the occurrence of anti-phospholipid and anti-beta2-GPI activities separately on heavy and light chains of an autoantibody. The possible mechanisms that such antibodies may employ to recognise their antigens, are discussed.

  12. Monoclonal antibodies against goldfish (Carassius auratus) immunoglobulin: application to the quantification of immunoglobulin and antibody-secreting cells by ELISPOT and seric immunoglobulin and antibody levels by ELISA in carp (Cyprinus carpio).

    PubMed

    Siwicki, A K; Vergnet, C; Charlemagne, J; Dunier, M

    1994-01-01

    Monoclonal antibodies (mAbs) raised against heavy and light chains of goldfish immunoglobulin (Ig) were characterized by a Western blot technique. A complete cross-reactivity was observed between carp and goldfish Ig. These mAbs were used for the quantification of carp Ig and anti-Yersinia ruckeri antibodies by ELISA. An ELISPOT assay was also developed in carp to quantify Ig-secreting cells (ISC) and antibody-secreting cells (ASC). The number of ASC was maximum on day 18 post-vaccination and decreased to the basal level on day 28. The antibody levels in sera were maximum on day 18 and slowly decreased until day 28. PMID:7951348

  13. Neuregulin1-β decreases interleukin-1β-induced RhoA activation, myosin light chain phosphorylation, and endothelial hyperpermeability.

    PubMed

    Wu, Limin; Ramirez, Servio H; Andrews, Allison M; Leung, Wendy; Itoh, Kanako; Wu, Jiang; Arai, Ken; Lo, Eng H; Lok, Josephine

    2016-01-01

    Neuregulin-1 (NRG1) is an endogenous growth factor with multiple functions in the embryonic and postnatal brain. The NRG1 gene is large and complex, transcribing more than twenty transmembrane proteins and generating a large number of isoforms in tissue and cell type-specific patterns. Within the brain, NRG1 functions have been studied most extensively in neurons and glia, as well as in the peripheral vasculature. Recently, NRG1 signaling has been found to be important in the function of brain microvascular endothelial cells, decreasing IL-1β-induced increases in endothelial permeability. In the current experiments, we have investigated the pathways through which the NRG1-β isoform acts on IL-1β-induced endothelial permeability. Our data show that NRG1-β increases barrier function, measured by transendothelial electrical resistance, and decreases IL-1β-induced hyperpermeability, measured by dextran-40 extravasation through a monolayer of brain microvascular endothelial cells plated on transwells. An investigation of key signaling proteins suggests that the effect of NRG1-β on endothelial permeability is mediated through RhoA activation and myosin light chain phosphorylation, events which affect filamentous actin morphology. In addition, AG825, an inhibitor of the erbB2-associated tyrosine kinase, reduces the effect of NRG1-β on IL-1β-induced RhoA activation and myosin light chain phosphorylation. These data add to the evidence that NRG1-β signaling affects changes in the brain microvasculature in the setting of neuroinflammation. We propose the following events for neuregulin-1-mediated effects on Interleukin-1 β (IL-1β)-induced endothelial hyperpermeability: IL-1β leads to RhoA activation, resulting in an increase in phosphorylation of myosin light chain (MLC). Phosphorylation of MLC is known to result in actin contraction and alterations in the f-actin cytoskeletal structure. These changes are associated with increased endothelial permeability

  14. Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex

    SciTech Connect

    Silvaggi,N.; Wilson, D.; Tzipori, S.; Allen, K.

    2008-01-01

    The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 Angstroms-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S? atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-bound structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 Angstroms resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.

  15. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.

    PubMed

    Lima, V V; Lobato, N S; Filgueira, F P; Webb, R C; Tostes, R C; Giachini, F R

    2014-10-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction. PMID:25140811

  16. Effect of nucleotides and actin on the orientation of the light chain-binding domain in myosin subfragment 1.

    PubMed

    Smyczynski, C; Kasprzak, A A

    1997-10-28

    The X-ray structure of myosin head (S1) reveals the presence of a long alpha-helical structure that supports both the essential and the regulatory light chains. It has been proposed that small structural changes in the catalytic domain of S1 are amplified by swinging the long alpha-helix (the "lever arm") to produce approximately 11 nm steps. To probe the spatial position of the putative lever in various S1 states, we have measured, by fluorescence resonance energy transfer (FRET), the effect of nucleotides and actin on the distances between Cys-177 of the essential light chain A1 (which is attached to the alpha-helix) and three loci in the catalytic domain. Cys-177 (donor) was labeled with 1,5-IAEDANS. The trinitrophenylated ADP analog (TNP-ADP, acceptor) was used to measure the distance to the active site. Lys-553 at the actin-binding site, labeled with a fluorescein derivative, and Lys-83 modified with trinitrobenzenesulfonic acid served as two other acceptors. FRET measurements were performed for S1 alone, for its complexes with MgADP and MgATP, for the analogs of the transition state of the ATPase reaction, S1.ADP.BeFx, S1.ADP.AlF4, and S1.ADP.VO4, and for acto-S1 in the absence and in the presence of ADP. When the transition state and acto-S1 complexes were formed, the change in the Cys-177 --> Lys-83 distance was <1.1 A, for the distance Cys-177 --> Lys-553, the change was +/-2.5 A. These distance changes correspond to rotations by <10 degrees and approximately 25 degrees, respectively. For the Cys-177 --> TNP-ADP the interprobe separation decreased by approximately 6 A in the presence of BeFx and AlF4- but only 1.9 A in the presence of vanadate; we do not interpret the 6 A change as resulting from the lever rotation. Using the coordinates of the acto-S1 complex, we have computed the expected changes in these distances resulting from rotation of the lever. These changes were much greater than the ones observed. The above results are inconsistent with models

  17. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.

    PubMed

    Lima, V V; Lobato, N S; Filgueira, F P; Webb, R C; Tostes, R C; Giachini, F R

    2014-10-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

  18. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

    PubMed Central

    Lima, V.V.; Lobato, N.S.; Filgueira, F.P.; Webb, R.C.; Tostes, R.C.; Giachini, F.R.

    2014-01-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction. PMID:25140811

  19. Induction of light chain replacement in human plasma cells by caffeine is independent from both the upregulation of RAG protein expression and germ line transcription.

    PubMed

    Tachibana, H; Haruta, H; Ueda, K; Chiwata, T; Yamada, K

    2000-02-25

    When some human plasma cell lines are cultured with concanavalin A, the original light chain is replaced with another light chain which results from secondary VJ recombination (light chain shifting). We examined various intracellular factors involved in the induction of light chain shifting. Light chain shifting can be induced upon treatment with agents with phosphatase inhibitory activity such as caffeine and okadaic acid. Although the plasma cells used express both RAG-1 and RAG-2, the expression level of these proteins was not affected by caffeine or okadaic acid. Transcription of the germ line locus, which correlates to the locus activation for rearrangement, is also not influenced by phosphatase inhibition. However, the amount of signal broken-ended DNA intermediates generated during V(D)J rearrangement was shown to increase upon caffeine or okadaic acid treatment. The inhibitory activity of caffeine on phosphatase was the same as okadaic acid. However, caffeine exhibited much higher activity for VJ coding joint formation than okadaic acid. Therefore, although phosphatase inhibition might act, in part, on a mechanism by which V(D)J recombinase activity is regulated within the human plasma cells, other factor(s) are probably also involved in the process.

  20. Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody.

    PubMed

    Vlasak, Josef; Bussat, Marie C; Wang, Shiyi; Wagner-Rousset, Elsa; Schaefer, Mark; Klinguer-Hamour, Christine; Kirchmeier, Marc; Corvaïa, Nathalie; Ionescu, Roxana; Beck, Alain

    2009-09-15

    Despite technological advances, detection of deamidation in large proteins remains a challenge and the use of orthogonal methods is needed for unequivocal assignment. By a combination of cation-exchange separation, papain digestion, and a panel of mass spectrometry techniques we identified asparagine deamidation in light chain complementarity determining region 1 (CDR1) of a humanized IgG1 monoclonal antibody. The reaction yields both Asp and isoAsp, which were assigned by Edman degradation and by isoAsp detection using protein isoaspartate methyltransferase. The deamidated antibody variants were less potent in antigen binding compared to the nondegraded antibody. Changes in near-UV CD spectra, susceptibility to papain cleavage in an adjacent CDR2 loop, and the tendency of the newly formed isoAsp to undergo isomerization suggest local perturbations in the structure of the isoAsp-containing antibody.

  1. Cardiac myosin light chain phosphorylation and inotropic effects of a biased ligand, TRV120023, in a dilated cardiomyopathy model

    PubMed Central

    Tarigopula, Madhusudhan; Davis, Robert T.; Mungai, Paul T.; Ryba, David M.; Wieczorek, David F.; Cowan, Conrad L.; Violin, Jonathan D.; Wolska, Beata M.; Solaro, R. John

    2015-01-01

    Aims Therapeutic approaches to treat familial dilated cardiomyopathy (DCM), which is characterized by depressed sarcomeric tension and susceptibility to Ca2+-related arrhythmias, have been generally unsuccessful. Our objective in the present work was to determine the effect of the angiotensin II type 1 receptor (AT1R) biased ligand, TRV120023, on contractility of hearts of a transgenic mouse model of familial DCM with mutation in tropomyosin at position 54 (TG-E54K). Our rationale is based on previous studies, which have supported the hypothesis that biased G-protein-coupled receptor ligands, signalling via β-arrestin, increase cardiac contractility with no effect on Ca2+ transients. Our previous work demonstrated that the biased ligand TRV120023 is able to block angiotensin-induced hypertrophy, while promoting an increase in sarcomere Ca2+ response. Methods and results We tested the hypothesis that the depression in cardiac function associated with DCM can be offset by infusion of the AT1R biased ligand, TRV120023. We intravenously infused saline, TRV120023, or the unbiased ligand, losartan, for 15 min in TG-E54K and non-transgenic mice to obtain left ventricular pressure–volume relations. Hearts were analysed for sarcomeric protein phosphorylation. Results showed that the AT1R biased ligand increases cardiac performance in TG-E54K mice in association with increased myosin light chain-2 phosphorylation. Conclusion Treatment of mice with an AT1R biased ligand, acting via β-arrestin signalling, is able to induce an increase in cardiac contractility associated with an increase in ventricular myosin light chain-2 phosphorylation. AT1R biased ligands may prove to be a novel inotropic approach in familial DCM. PMID:26045475

  2. Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

    PubMed Central

    Wong, Raymond; Fabian, Lacramioara; Forer, Arthur; Brill, Julie A

    2007-01-01

    Background Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated. Results Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3. Conclusion We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring. PMID:17509155

  3. A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring 6 Light-Chain Fibrillogenesis

    SciTech Connect

    Hernández-Santoyo, A.; Del Pozo Yauner, L; Fuentes-Silva, D; Ortiz, E; Rudiño-Piñera, E; Sánchez-López, R; Horjales, E; Becerril, B; Rodríguez-Romero, A

    2010-01-01

    Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although {lambda} chains, particularly those belonging to the {lambda}6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the {lambda}6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the VL (variable region of the light chain)-VL interface. This mutant crystallized in two orthorhombic polymorphs, P2{sub 1}2{sub 1}2{sub 1} and C222{sub 1}. In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222{sub 1} lattice showed the establishment of intermolecular {beta}-{beta} interactions that involved the N-terminus and {beta}-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the VL interface in {lambda}6 LCs.

  4. Anti-nociceptive effect of a conjugate of substance P and light chain of botulinum neurotoxin type A.

    PubMed

    Mustafa, Golam; Anderson, Ethan M; Bokrand-Donatelli, Yvonne; Neubert, John K; Caudle, Robert M

    2013-11-01

    Neuropathic pain is a debilitating condition resulting from damage to sensory transmission pathways in the peripheral and central nervous system. A potential new way of treating chronic neuropathic pain is to target specific pain-processing neurons based on their expression of particular receptor molecules. We hypothesized that a toxin-neuropeptide conjugate would alter pain by first being taken up by specific receptors for the neuropeptide expressed on the neuronal cells. Then, once inside the cell the toxin would inhibit the neurons' activity without killing the neurons, thereby providing pain relief without lesioning the nervous system. In an effort to inactivate the nociceptive neurons in the trigeminal nucleus caudalis in mice, we targeted the NK1 receptor (NK1R) using substance P (SP). The catalytically active light chain of botulinum neurotoxin type A (LC/A) was conjugated with SP. Our results indicate that the conjugate BoNT/A-LC:SP is internalized in cultured NK1R-expressing neurons and also cleaves the target of botulinum toxin, a component-docking motif necessary for release of neurotransmitters called SNAP-25. The conjugate was next tested in a murine model of Taxol-induced neuropathic pain. An intracisternal injection of BoNT/A-LC:SP decreased thermal hyperalgesia as measured by the operant orofacial nociception assay. These findings indicate that conjugates of the light chain of botulinum toxin are extremely promising agents for use in suppressing neuronal activity for extended time periods, and that BoNT/A-LC:SP may be a useful agent for treating chronic pain.

  5. Low cost omega navigation receiver

    NASA Technical Reports Server (NTRS)

    Lilley, R. W.

    1974-01-01

    The development of a low cost Omega navigation receiver is discussed. Emphasis is placed on the completion and testing of a modular, multipurpose Omega receiver which utilizes a digital memory-aided, phase-locked loop to provide phase measurement data to a variety of applications interfaces. The functional units contained in the prototype device are described. The receiver is capable of receiving and storing phase measurements for up to eight Omega signals and computes two switch-selectable lines of position, displaying this navigation data in chart-recorded form.

  6. Omega-3 fats: Good for your heart

    MedlinePlus

    ... arteries - omega-3s; Coronary artery disease - omega-3s; Heart disease - omega-3s ... Omega-3s are good for your heart and blood vessels in several ways. They reduce triglycerides , a type of fat in your blood. They reduce the risk of an ...

  7. Evidence indicating independent assortment of framework and complementarity-determining segments of the variable regions of rabbit light chains. Delineation of a possible J minigene.

    PubMed

    Kabat, E A; Wu, T T; Bilofsky, H

    1980-07-01

    Amino acid sequences of rabbit light chains show considerable evidence of independent assortment of framework (FR) and complementarity-determining (CDR) segments. This suggests that they are coded for by independent genetic units (minigaenes) and that individual light chains are assembled somatically by recombining these units. Identical FR sets with multiple members generally comprise chains with different specificities, whereas identical CDR sets tend to have chains of a single specificity. A J segment, which, by analogy with mouse light chains, is made up of the last two residues of CDR3 plus all of FR4, contained 18 different sets and could contribute to diversity generated by CDR3. The longest segment, FR3, had a very large number of sets. Evidence is presented showing that the number of sets could be substantially reduced by permitting FR3 to be formed by two independently assorting segments comprising residues 57-68 and 69-88.

  8. A human Fab fragment specific for thyroid peroxidase generated by cloning thyroid lymphocyte-derived immunoglobulin genes in a bacteriophage lambda library.

    PubMed

    Portolano, S; Seto, P; Chazenbalk, G D; Nagayama, Y; McLachlan, S M; Rapoport, B

    1991-08-30

    A human Fab fragment (SP2) which binds specifically to human thyroid peroxidase has been generated by expressing random combinations of heavy and light chain immunoglobulin genes (derived from Graves' thyroid cDNA) in a bacteriophage lambda library. In common with many serum TPO autoantibodies, the cloned Fab fragment is IgG1 kappa and has a high affinity for TPO (approximately 10(-9) M). On the basis of their nucleotide sequences, the heavy and light chain genes coding for SP2 belong to families VHI, (D), JH3 and VKI, JK2, respectively. These data provide the first characterization at a molecular level of a human thyroid peroxidase antibody associated with autoimmune thyroid disease.

  9. Immunoglobulin genes and antibody responses in the spotted wolffish (Anarhichas minor Olafsen).

    PubMed

    Espelid, S; Halse, M; Solem, S T; Jørgensen, T O

    2001-07-01

    The spotted wolffish Anarhichas minor Olafsen is a promising new species in aquaculture in the cold waters of northern Norway. In this paper, some basic immunological studies of this marine species are reported. Of comparative interest are the cDNA sequences of the immunoglobulin transcript and the antibody responses to model antigens. Of more practical importance are the humoral immune responses and antibody specificities to potentially pathogenic bacteria. Full length cDNA clones encoding the immunoglobulin heavy and light chains in the spotted wolffish were sequenced demonstrating variable degrees of similarity to other teleost fish species. Also in the spotted wolffish the CH4 domain was deleted in the transmembrane form of the immunoglobulin heavy chain (IgH) as a receptor on B cells, with the transmembrane exon spliced directly to the CH3 domain. The antibody responses to various antigens like hapten-carrier molecules, protein antigens and bacterial pathogens were relatively high, but with some interesting exceptions. Anti-hapten responses to NIP and FITC were high while anti-DNS responses were low, but more surprisingly, there was hardly any B-cell response to the carrier molecule LPH. On the other hand, protein antigens like CGG and BSA were highly immunogenic in the spotted wolffish as were the bacterial antigens Vibrio anguillarum, V. salmonicida and Aeromonas salmonicida.

  10. Short Zoom Into Omega Centauri

    NASA Video Gallery

    This is a zoom into a simulated model of the globular star cluster Omega Centauri. All the stars appear to be moving in random directions, like a swarm of bees. Astronomers used Hubble's exquisite ...

  11. Light Chain Deposition Disease in an Older Adult Patient Successfully Treated with Long-term Administration of Bortezomib, Melphalan and Prednisone.

    PubMed

    Hiyamuta, Hiroto; Yamada, Shunsuke; Matsukuma, Yuta; Tsuchimoto, Akihiro; Nakano, Toshiaki; Taniguchi, Masatomo; Masutani, Kosuke; Yoshimoto, Goichi; Muta, Tsuyoshi; Akashi, Koichi; Kitazono, Takanari; Tsuruya, Kazuhiko

    2016-01-01

    A 70-year-old woman was admitted to our hospital because of fatigue and renal dysfunction and was diagnosed with light chain deposition disease (LCDD) with multiple organ involvement (kidney, thyroid gland, heart and eyes). After chemotherapy with bortezomib, cyclophosphamide and dexamethasone, hepatobiliary enzyme levels increased abruptly. A liver biopsy showed light chain deposition in Disse spaces. After two years of treatment with bortezomib, melphalan and prednisone (VMP) administered at shorter intervals relative to regular cycles, the patient showed a hematological and organ response. This case indicates that a relatively low dose intensity VMP regimen is preferable for elderly patients with LCDD with multiple organ involvement. PMID:27181540

  12. Triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production.

    PubMed

    Matsushita, Hiroaki; Sano, Akiko; Wu, Hua; Jiao, Jin-An; Kasinathan, Poothappillai; Sullivan, Eddie J; Wang, Zhongde; Kuroiwa, Yoshimi

    2014-01-01

    Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML1-/-; double knockouts or DKO). However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK) and lambda-chain (bIGL) genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ) and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ). To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J) and constant (C) gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5) by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM-/-, bIGHML1-/- and bIGL-/-; TKO) Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ).

  13. Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production

    PubMed Central

    Matsushita, Hiroaki; Sano, Akiko; Wu, Hua; Jiao, Jin-an; Kasinathan, Poothappillai; Sullivan, Eddie J.; Wang, Zhongde; Kuroiwa, Yoshimi

    2014-01-01

    Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM−/−, bIGHML1−/−; double knockouts or DKO). However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK) and lambda-chain (bIGL) genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ) and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ). To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J) and constant (C) gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5) by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM−/−, bIGHML1−/− and bIGL−/−; TKO) Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ). PMID:24603704

  14. Clonal deletion of specific thymocytes by an immunoglobulin idiotype.

    PubMed Central

    Bogen, B; Dembic, Z; Weiss, S

    1993-01-01

    We have investigated whether immunoglobulin can induce clonal deletion of thymocytes by employing two strains of transgenic mice. One strain is transgenic for an alpha/beta T cell receptor (TCR) which recognizes a processed idiotypic peptide of the lambda 2(315) light chain variable region, bound to the I-Ed class II major histocompatibility complex molecule. The other mouse strain is transgenic for the lambda 2(315) gene. Double transgenic offspring from a TCR-transgenic female mated with a lambda 2(315) transgenic male exhibit a pronounced clonal deletion of CD4+CD8+ thymocytes. Analysis of neonates from the reciprocal (lambda 2(315)-transgenic female x TCR-transgenic male) cross suggests that the deletion in double transgenic offspring most likely is caused by lambda 2(315) produced within the thymus rather than by maternally derived IgG, lambda 2(315). Nevertheless, IgG, lambda 2(315) can cause deletion of CD4+CD8+ thymocytes when injected in large amounts intraperitoneally into either adult or neonatal TCR-transgenic mice. Deletion is evident 48 and 72 h after injection, but by day 7 the thymus has already regained its normal appearance. A serum concentration of several hundred microgram/ml is required for deletion to be observed. Therefore, the heterogeneous idiotypes of serum Ig are probably each of too low concentration to cause thymocyte deletion in normal animals. Images PMID:8428591

  15. Immunoglobulin genomics in the prairie vole (Microtus ochrogaster).

    PubMed

    Qin, Tong; Zhao, Huijing; Zhu, Huabin; Wang, Dong; Du, Weihua; Hao, Haisheng

    2015-08-01

    In science, the prairie voles are ideal models for studying the regulatory mechanisms of social behavior in humans. The utility of the prairie vole as a biology model can be further enhanced by characterization of the genes encoding components of the immune system. Here, we report the genomic organization of the prairie vole immunoglobulin heavy and light chain genes. The prairie vole IgH locus on chromosome 1 spans over 1600kb, and consists of at least 79 VH segments (28 potentially functional genes, 2 ORFs and 49 pseudogenes), 7 DH segments, 4 JH segments, four constant region genes (μ, γ, ɛ, and α), and two transmembrane regions of δ gene. The Igκ locus, found on three scaffolds (JH996430, JH996605 and JH996566), contains a totle of 124 Vκ segments (47 potentially functional genes, 1 ORF and 76 pseudogenes), 5 Jκ segments and a single Cκ gene. Two different transcriptional orientations were determined for these Vκ gene segments. In contrast, the Igλ locus on scaffold JH996473 and JH996489 includes 21 Vλ gene segments (14 potentially functional genes, 1 ORF and 6 pseudogenes), all with the same transcriptional polarity as the downstream Jλ-Cλ cluster. Phylogenetic analysis and sequence alignments suggested the prairie vole's large germline VH, Vκ and Vλ gene segments appear to form limited gene families. Therefore, this species may generate antibody diversity via a gene conversion-like mechanism associated with its pseudogene reserves.

  16. Immunoglobulin genomics in the prairie vole (Microtus ochrogaster).

    PubMed

    Qin, Tong; Zhao, Huijing; Zhu, Huabin; Wang, Dong; Du, Weihua; Hao, Haisheng

    2015-08-01

    In science, the prairie voles are ideal models for studying the regulatory mechanisms of social behavior in humans. The utility of the prairie vole as a biology model can be further enhanced by characterization of the genes encoding components of the immune system. Here, we report the genomic organization of the prairie vole immunoglobulin heavy and light chain genes. The prairie vole IgH locus on chromosome 1 spans over 1600kb, and consists of at least 79 VH segments (28 potentially functional genes, 2 ORFs and 49 pseudogenes), 7 DH segments, 4 JH segments, four constant region genes (μ, γ, ɛ, and α), and two transmembrane regions of δ gene. The Igκ locus, found on three scaffolds (JH996430, JH996605 and JH996566), contains a totle of 124 Vκ segments (47 potentially functional genes, 1 ORF and 76 pseudogenes), 5 Jκ segments and a single Cκ gene. Two different transcriptional orientations were determined for these Vκ gene segments. In contrast, the Igλ locus on scaffold JH996473 and JH996489 includes 21 Vλ gene segments (14 potentially functional genes, 1 ORF and 6 pseudogenes), all with the same transcriptional polarity as the downstream Jλ-Cλ cluster. Phylogenetic analysis and sequence alignments suggested the prairie vole's large germline VH, Vκ and Vλ gene segments appear to form limited gene families. Therefore, this species may generate antibody diversity via a gene conversion-like mechanism associated with its pseudogene reserves. PMID:26073565

  17. Characterization of omega-3 tablets.

    PubMed

    Vestland, Tina Lien; Jacobsen, Øyvind; Sande, Sverre Arne; Myrset, Astrid Hilde; Klaveness, Jo

    2016-04-15

    Omega-3 nutraceuticals are extensively used as health supplements worldwide. Various administration forms for delivery of omega-3 are available. However, the niche omega-3 tablets have so far remained unexplored. In this work tablets containing 25-40% (w/w) omega-3 oil as triglycerides or ethyl esters were prepared utilizing a direct compaction grade powder with β-cyclodextrin as encapsulating agent. It was found that powders with up to 35% (w/w) triglyceride oil and 30% (w/w) ethyl ester oil, respectively, can be directly compressed into tablets of excellent quality. Physical properties of omega-3 containing powders and tablets are described. The powder X-ray diffractograms of the powders and crushed tablets show evidence of the formation of new crystalline phases not present in β-cyclodextrin. In addition, (1)H NMR data suggest that the ethyl esters form inclusion complexes with β-cyclodextrin. Compaction of other, commercially available, omega-3 powders was performed as a comparison and deemed unsuccessful. PMID:26616980

  18. Characterization of omega-3 tablets.

    PubMed

    Vestland, Tina Lien; Jacobsen, Øyvind; Sande, Sverre Arne; Myrset, Astrid Hilde; Klaveness, Jo

    2016-04-15

    Omega-3 nutraceuticals are extensively used as health supplements worldwide. Various administration forms for delivery of omega-3 are available. However, the niche omega-3 tablets have so far remained unexplored. In this work tablets containing 25-40% (w/w) omega-3 oil as triglycerides or ethyl esters were prepared utilizing a direct compaction grade powder with β-cyclodextrin as encapsulating agent. It was found that powders with up to 35% (w/w) triglyceride oil and 30% (w/w) ethyl ester oil, respectively, can be directly compressed into tablets of excellent quality. Physical properties of omega-3 containing powders and tablets are described. The powder X-ray diffractograms of the powders and crushed tablets show evidence of the formation of new crystalline phases not present in β-cyclodextrin. In addition, (1)H NMR data suggest that the ethyl esters form inclusion complexes with β-cyclodextrin. Compaction of other, commercially available, omega-3 powders was performed as a comparison and deemed unsuccessful.

  19. Nutraceutical with Resveratrol and Omega-3 Fatty Acids Induces Autophagy in ARPE-19 Cells.

    PubMed

    Koskela, Ali; Reinisalo, Mika; Petrovski, Goran; Sinha, Debasish; Olmiere, Céline; Karjalainen, Reijo; Kaarniranta, Kai

    2016-05-11

    Impaired autophagic and proteasomal cleansing have been documented in aged retinal pigment epithelial (RPE) cells and age-related macular degeneration (AMD). Omega-3 fatty acids and resveratrol have many positive homeostatic effects in RPE cells. In this work, ARPE-19 cells were treated with 288 ng of Resvega, containing 30 mg of trans resveratrol and 665 mg of omega-3 fatty acids, among other nutrients, with proteasome inhibitor MG-132 or autophagy inhibitor bafilomycin A1 up to 48 h. Autophagy markers p62/SQSTM1 (p62) and LC3 (microtubule-associated protein 1A/1B-light chain 3) were analyzed by Western blotting. Fluorescence microscopy with mCherry-GFP-LC3 plasmid was applied to study the autophagy flux, and cytoprotective effects were investigated with colorimetric MTT and LDH assays. Resvega induced autophagy by showing increased autolysosome formation and autophagy flux, and the change in the p62 and LC3 protein levels further confirmed the fluorescent microscopy results. Moreover, Resvega provided a clear cytoprotection under proteasome inhibition. These findings highlight the potential of the nutraceuticals containing resveratrol, omega-3 fatty acids and other nutrients in the prevention of ARPE-19 cell damage.

  20. Nutraceutical with Resveratrol and Omega-3 Fatty Acids Induces Autophagy in ARPE-19 Cells.

    PubMed

    Koskela, Ali; Reinisalo, Mika; Petrovski, Goran; Sinha, Debasish; Olmiere, Céline; Karjalainen, Reijo; Kaarniranta, Kai

    2016-01-01

    Impaired autophagic and proteasomal cleansing have been documented in aged retinal pigment epithelial (RPE) cells and age-related macular degeneration (AMD). Omega-3 fatty acids and resveratrol have many positive homeostatic effects in RPE cells. In this work, ARPE-19 cells were treated with 288 ng of Resvega, containing 30 mg of trans resveratrol and 665 mg of omega-3 fatty acids, among other nutrients, with proteasome inhibitor MG-132 or autophagy inhibitor bafilomycin A1 up to 48 h. Autophagy markers p62/SQSTM1 (p62) and LC3 (microtubule-associated protein 1A/1B-light chain 3) were analyzed by Western blotting. Fluorescence microscopy with mCherry-GFP-LC3 plasmid was applied to study the autophagy flux, and cytoprotective effects were investigated with colorimetric MTT and LDH assays. Resvega induced autophagy by showing increased autolysosome formation and autophagy flux, and the change in the p62 and LC3 protein levels further confirmed the fluorescent microscopy results. Moreover, Resvega provided a clear cytoprotection under proteasome inhibition. These findings highlight the potential of the nutraceuticals containing resveratrol, omega-3 fatty acids and other nutrients in the prevention of ARPE-19 cell damage. PMID:27187449

  1. Nutraceutical with Resveratrol and Omega-3 Fatty Acids Induces Autophagy in ARPE-19 Cells

    PubMed Central

    Koskela, Ali; Reinisalo, Mika; Petrovski, Goran; Sinha, Debasish; Olmiere, Céline; Karjalainen, Reijo; Kaarniranta, Kai

    2016-01-01

    Impaired autophagic and proteasomal cleansing have been documented in aged retinal pigment epithelial (RPE) cells and age-related macular degeneration (AMD). Omega-3 fatty acids and resveratrol have many positive homeostatic effects in RPE cells. In this work, ARPE-19 cells were treated with 288 ng of Resvega, containing 30 mg of trans resveratrol and 665 mg of omega-3 fatty acids, among other nutrients, with proteasome inhibitor MG-132 or autophagy inhibitor bafilomycin A1 up to 48 h. Autophagy markers p62/SQSTM1 (p62) and LC3 (microtubule-associated protein 1A/1B-light chain 3) were analyzed by Western blotting. Fluorescence microscopy with mCherry-GFP-LC3 plasmid was applied to study the autophagy flux, and cytoprotective effects were investigated with colorimetric MTT and LDH assays. Resvega induced autophagy by showing increased autolysosome formation and autophagy flux, and the change in the p62 and LC3 protein levels further confirmed the fluorescent microscopy results. Moreover, Resvega provided a clear cytoprotection under proteasome inhibition. These findings highlight the potential of the nutraceuticals containing resveratrol, omega-3 fatty acids and other nutrients in the prevention of ARPE-19 cell damage. PMID:27187449

  2. Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

    SciTech Connect

    Colson, Brett A.; Locher, Matthew R.; Bekyarova, Tanya; Patel, Jitandrakumar R.; Fitzsimons, Daniel P.; Irving, Thomas C.; Moss, Richard L.

    2010-05-25

    Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca{sup 2+} sensitivity of force (pCa{sub 50}), PKA treatment has been shown to decrease pCa{sub 50}, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca{sup 2+}-independent force and maximum Ca{sup 2+}-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I{sub 1,1}/I{sub 1,0}) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d{sub 1,0}) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I{sub 1,1}/I{sub 1,0} and, as hypothesized, treatment with MLCK also increased I{sub 1,1}/I{sub 1,0}, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by {approx}2 nm ({Delta} 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca{sup 2+} sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

  3. Spatial profile reconstruction of individual componentsof the nonlinear susceptibility tensors {chi}-circumflex {sup (3)}(z, {omega}', {omega}' -{omega}, {omega}) and {chi}-circumflex {sup (3)}(z, 2{omega}{+-}{omega}', {+-}{omega}', {omega}, {omega}) of a one-dimensionally inhomogeneous medium

    SciTech Connect

    Golubkov, A A; Makarov, Vladimir A

    2011-06-30

    We have proved for the first time and proposed an algorithm of unique spatial profile reconstruction of the components {chi}-circumflex {sup (3)}{sub yyyy} of complex tensors {chi}-circumflex {sup (3)}(z, {omega}', {omega}', -{omega}, {omega}) and {chi}-circumflex {sup (3)}(z, 2{omega}{+-}{omega}', {+-}{omega}', {omega}, {omega}), describing four-photon interaction of light waves in a one-dimensionally inhomogeneous plate, whose medium has a symmetry plane m{sub y} that is perpendicular to its surface. For the media with an additional symmetry axis 2{sub z}, 4{sub z}, 6{sub z} or {infinity}{sub z} that is perpendicular to the plate surface, the proposed method can be used to reconstruct about one-fifth of all independent components of the above tensors. (nonlinear optical phenomena)

  4. Mining the antibodyome for HIV-1-neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains.

    PubMed

    Zhu, Jiang; Ofek, Gilad; Yang, Yongping; Zhang, Baoshan; Louder, Mark K; Lu, Gabriel; McKee, Krisha; Pancera, Marie; Skinner, Jeff; Zhang, Zhenhai; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E; Blinn, Julie; Alam, S Munir; Haynes, Barton F; Simek, Melissa; Burton, Dennis R; Koff, Wayne C; Mullikin, James C; Mascola, John R; Shapiro, Lawrence; Kwong, Peter D

    2013-04-16

    Next-generation sequencing of antibody transcripts from HIV-1-infected individuals with broadly neutralizing antibodies could provide an efficient means for identifying somatic variants and characterizing their lineages. Here, we used 454 pyrosequencing and identity/divergence grid sampling to analyze heavy- and light-chain sequences from donor N152, the source of the broadly neutralizing antibody 10E8. We identified variants with up to 28% difference in amino acid sequence. Heavy- and light-chain phylogenetic trees of identified 10E8 variants displayed similar architectures, and 10E8 variants reconstituted from matched and unmatched phylogenetic branches displayed significantly lower autoreactivity when matched. To test the generality of phylogenetic pairing, we analyzed donor International AIDS Vaccine Initiative 84, the source of antibodies PGT141-145. Heavy- and light-chain phylogenetic trees of PGT141-145 somatic variants also displayed remarkably similar architectures; in this case, branch pairings could be anchored by known PGT141-145 antibodies. Altogether, our findings suggest that phylogenetic matching of heavy and light chains can provide a means to approximate natural pairings.

  5. [The effect of phosphorylation of myosin light chains on the structural state of tropomyosin in thin filaments, decorated with heavy meromyosin].

    PubMed

    Vorovikov, Iu S; Szczesna, D; Kakol, I

    1989-06-01

    The structural state of tropomyosin (TM) modified by 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonate (1.5-IAEDANS) upon F-actin decoration with myosin subfragment 1 (S1) and heavy meromyosin (HMM) in glycerinated myosin- and troponin-free muscle fibers was studied. HMM preparations contained native phosphorylated myosin light chains, while S1 preparations did not. The changes in the polarized fluorescence of 1.5-IAEDANS-TM during the F-actin interaction with S1 were independent of light chains phosphorylation and Ca2+ concentration, but were dependent on these factors during the F-actin interaction with HMM. The binding of myosin heads to F-actin is supposed to initiate conformational changes in TM which are accompanied by changes in the flexibility and molecular arrangement of TM. In the presence of light chains, the structural changes in TM depend on light chains phosphorylation and Ca2+ concentration. The conformational changes in TM seem to be responsible for the mechanisms of coupling of the myosin and tropomyosin modulation system during the actin-myosin interaction in skeletal muscles.

  6. omega-Helices in proteins.

    PubMed

    Enkhbayar, Purevjav; Boldgiv, Bazartseren; Matsushima, Norio

    2010-05-01

    A modification of the alpha-helix, termed the omega-helix, has four residues in one turn of a helix. We searched the omega-helix in proteins by the HELFIT program which determines the helical parameters-pitch, residues per turn, radius, and handedness-and p = rmsd/(N - 1)(1/2) estimating helical regularity, where "rmsd" is the root mean square deviation from the best fit helix and "N" is helix length. A total of 1,496 regular alpha-helices 6-9 residues long with p < or = 0.10 A were identified from 866 protein chains. The statistical analysis provides a strong evidence that the frequency distribution of helices versus n indicates the bimodality of typical alpha-helix and omega-helix. Sixty-two right handed omega-helices identified (7.2% of proteins) show non-planarity of the peptide groups. There is amino acid preference of Asp and Cys. These observations and analyses insist that the omega-helices occur really in proteins.

  7. omega-Helices in proteins.

    PubMed

    Enkhbayar, Purevjav; Boldgiv, Bazartseren; Matsushima, Norio

    2010-05-01

    A modification of the alpha-helix, termed the omega-helix, has four residues in one turn of a helix. We searched the omega-helix in proteins by the HELFIT program which determines the helical parameters-pitch, residues per turn, radius, and handedness-and p = rmsd/(N - 1)(1/2) estimating helical regularity, where "rmsd" is the root mean square deviation from the best fit helix and "N" is helix length. A total of 1,496 regular alpha-helices 6-9 residues long with p < or = 0.10 A were identified from 866 protein chains. The statistical analysis provides a strong evidence that the frequency distribution of helices versus n indicates the bimodality of typical alpha-helix and omega-helix. Sixty-two right handed omega-helices identified (7.2% of proteins) show non-planarity of the peptide groups. There is amino acid preference of Asp and Cys. These observations and analyses insist that the omega-helices occur really in proteins. PMID:20496104

  8. Regulation of cell wall synthesis by the clathrin light chain is essential for viability in Schizosaccharomyces pombe.

    PubMed

    de León, Nagore; Sharifmoghadam, Mohammad Reza; Hoya, Marta; Curto, M-Ángeles; Doncel, Cristina; Valdivieso, M-Henar

    2013-01-01

    The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, β(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization. PMID:23977061

  9. A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability*

    PubMed Central

    Clayburgh, Daniel R.; Rosen, Shari; Witkowski, Edwina D.; Wang, Fengjun; Blair, Stephanie; Dudek, Steven; Garcia, Joe G. N.; Alverdy, John C.; Turner, Jerrold R.

    2005-01-01

    Activation of Na+-nutrient cotransport leads to increased tight junction permeability in intestinal absorptive (villus) enterocytes. This regulation requires myosin II regulatory light chain (MLC) phosphorylation mediated by MLC kinase (MLCK). We examined the spatiotemporal segregation of MLCK isoform function and expression along the crypt-villus axis and found that long MLCK, which is expressed as two alternatively spliced isoforms, accounts for 97 ± 4% of MLC kinase activity in interphase intestinal epithelial cells. Expression of the MLCK1 isoform is limited to well differentiated enterocytes, both in vitro and in vivo, and this expression correlates closely with development of Na+-nutrient cotransport-dependent tight junction regulation. Consistent with this role, MLCK1 is localized to the perijunctional actomyosin ring. Furthermore, specific knockdown of MLCK1 using siRNA reduced tight junction permeability in monolayers with active Na+-glucose cotransport, confirming a functional role for MLCK1. These results demonstrate unique physiologically relevant patterns of expression and subcellular localization for long MLCK isoforms and show that MLCK1 is the isoform responsible for tight junction regulation in absorptive enterocytes. PMID:15507455

  10. A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability.

    PubMed

    Clayburgh, Daniel R; Rosen, Shari; Witkowski, Edwina D; Wang, Fengjun; Blair, Stephanie; Dudek, Steven; Garcia, Joe G N; Alverdy, John C; Turner, Jerrold R

    2004-12-31

    Activation of Na(+)-nutrient cotransport leads to increased tight junction permeability in intestinal absorptive (villus) enterocytes. This regulation requires myosin II regulatory light chain (MLC) phosphorylation mediated by MLC kinase (MLCK). We examined the spatiotemporal segregation of MLCK isoform function and expression along the crypt-villus axis and found that long MLCK, which is expressed as two alternatively spliced isoforms, accounts for 97 +/- 4% of MLC kinase activity in interphase intestinal epithelial cells. Expression of the MLCK1 isoform is limited to well differentiated enterocytes, both in vitro and in vivo, and this expression correlates closely with development of Na(+)-nutrient cotransport-dependent tight junction regulation. Consistent with this role, MLCK1 is localized to the perijunctional actomyosin ring. Furthermore, specific knockdown of MLCK1 using siRNA reduced tight junction permeability in monolayers with active Na(+)-glucose cotransport, confirming a functional role for MLCK1. These results demonstrate unique physiologically relevant patterns of expression and subcellular localization for long MLCK isoforms and show that MLCK1 is the isoform responsible for tight junction regulation in absorptive enterocytes.

  11. Tarantula Myosin Free Head Regulatory Light Chain Phosphorylation Stiffens N-terminal Extension Releasing it and Blocking its Docking Back

    PubMed Central

    Alamo, Lorenzo; Li, Xiaochuan (Edward); Espinoza-Fonseca, L. Michel; Pinto, Antonio; Thomas, David D.; Lehman, William; Padrón, Raúl

    2015-01-01

    Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence lengths analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft in the relaxed state. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation. PMID:26038302

  12. Four things to know about myosin light chains as reporters for non-muscle myosin-2 dynamics in live cells.

    PubMed

    Heissler, Sarah M; Sellers, James R

    2015-02-01

    The interplay between non-muscle myosins-2 and filamentous actin results in cytoplasmic contractility which is essential for eukaryotic life. Concomitantly, there is tremendous interest in elucidating the physiological function and temporal localization of non-muscle myosin-2 in cells. A commonly used method to study the function and localization of non-muscle myosin-2 is to overexpress a fluorescent protein (FP)-tagged version of the regulatory light chain (RLC) which binds to the myosin-2 heavy chain by mass action. Caveats about this approach include findings from recent studies indicating that the RLC does not bind exclusively to the non-muscle myosin-2 heavy chain. Rather, it can also associate with the myosin heavy chains of several other classes as well as other targets than myosin. In addition, the presence of the FP moiety may compromise myosin's enzymatic and mechanical performance. This and other factors to be discussed in this commentary raise questions about the possible complications in using FP-RLC as a marker for the dynamic localization and regulatory aspects of non-muscle myosin-2 motor functions in cell biological experiments.

  13. Neurofilament Light Chain in Blood and CSF as Marker of Disease Progression in Mouse Models and in Neurodegenerative Diseases.

    PubMed

    Bacioglu, Mehtap; Maia, Luis F; Preische, Oliver; Schelle, Juliane; Apel, Anja; Kaeser, Stephan A; Schweighauser, Manuel; Eninger, Timo; Lambert, Marius; Pilotto, Andrea; Shimshek, Derya R; Neumann, Ulf; Kahle, Philipp J; Staufenbiel, Matthias; Neumann, Manuela; Maetzler, Walter; Kuhle, Jens; Jucker, Mathias

    2016-07-01

    A majority of current disease-modifying therapeutic approaches for age-related neurodegenerative diseases target their characteristic proteopathic lesions (α-synuclein, Tau, Aβ). To monitor such treatments, fluid biomarkers reflecting the underlying disease process are crucial. We found robust increases of neurofilament light chain (NfL) in CSF and blood in murine models of α-synucleinopathies, tauopathy, and β-amyloidosis. Blood and CSF NfL levels were strongly correlated, and NfL increases coincided with the onset and progression of the corresponding proteopathic lesions in brain. Experimental induction of α-synuclein lesions increased CSF and blood NfL levels, while blocking Aβ lesions attenuated the NfL increase. Consistently, we also found NfL increases in CSF and blood of human α-synucleinopathies, tauopathies, and Alzheimer's disease. Our results suggest that CSF and particularly blood NfL can serve as a reliable and easily accessible biomarker to monitor disease progression and treatment response in mouse models and potentially in human proteopathic neurodegenerative diseases. PMID:27292537

  14. Resonance Assignments and Secondary Structure Analysis of Dynein Light Chain 8 by Magic-angle Spinning NMR Spectroscopy

    SciTech Connect

    Sun, Shangjin; Butterworth, Andrew H.; Paramasivam, Sivakumar; Yan, Si; Lightcap, Christine M.; Williams, John C.; Polenova, Tatyana E.

    2011-08-04

    Dynein light chain LC8 is the smallest subunit of the dynein motor complex and has been shown to play important roles in both dynein-dependent and dynein-independent physiological functions via its interaction with a number of its binding partners. It has also been linked to pathogenesis including roles in viral infections and tumorigenesis. Structural information for LC8-target proteins is critical to understanding the underlying function of LC8 in these complexes. However, some LC8-target interactions are not amenable to structural characterization by conventional structural biology techniques owing to their large size, low solubility, and crystallization difficulties. Here, we report magic-angle spinning (MAS) NMR studies of the homodimeric apo-LC8 protein as a first effort in addressing more complex, multi-partner, LC8-based protein assemblies. We have established site-specific backbone and side-chain resonance assignments for the majority of the residues of LC8, and show TALOS+-predicted torsion angles ø and ψ in close agreement with most residues in the published LC8 crystal structure. Data obtained through these studies will provide the first step toward using MAS NMR to examine the LC8 structure, which will eventually be used to investigate protein–protein interactions in larger systems that cannot be determined by conventional structural studies.

  15. The effect of anti-inflammatory properties of ferritin light chain on lipopolysaccharide-induced inflammatory response in murine macrophages.

    PubMed

    Fan, Yumei; Zhang, Jie; Cai, Linlin; Wang, Shengnan; Liu, Caizhi; Zhang, Yongze; You, Linhao; Fu, Yujian; Shi, Zhenhua; Yin, Zhimin; Luo, Lan; Chang, Yanzhong; Duan, Xianglin

    2014-11-01

    Ferritin light chain (FTL) reduces the free iron concentration by forming ferritin complexes with ferritin heavy chain (FTH). Thus, FTL competes with the Fenton reaction by acting as an antioxidant. In the present study, we determined that FTL influences the lipopolysaccharide (LPS)-induced inflammatory response. FTL protein expression was regulated by LPS stimulation in RAW264.7 cells. To investigate the role of FTL in LPS-activated murine macrophages, we established stable FTL-expressing cells and used shRNA to silence FTL expression in RAW264.7 cells. Overexpression of FTL significantly decreased the LPS-induced production of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), nitric oxide (NO) and prostaglandin E2 (PGE2). Additionally, overexpression of FTL decreased the LPS-induced increase of the intracellular labile iron pool (LIP) and reactive oxygen species (ROS). Moreover, FTL overexpression suppressed the LPS-induced activation of MAPKs and nuclear factor-κB (NF-κB). In contrast, knockdown of FTL by shRNA showed the reverse effects. Therefore, our results indicate that FTL plays an anti-inflammatory role in response to LPS in murine macrophages and may have therapeutic potential for treating inflammatory diseases.

  16. Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction.

    PubMed

    Gregorich, Zachery R; Peng, Ying; Cai, Wenxuan; Jin, Yutong; Wei, Liming; Chen, Albert J; McKiernan, Susan H; Aiken, Judd M; Moss, Richard L; Diffee, Gary M; Ge, Ying

    2016-08-01

    Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia. PMID:27362462

  17. Utility of Serum Free Light Chain Measurements in Multiple Myeloma Patients Not Achieving Complete Response to Therapy

    PubMed Central

    Moustafa, Muhamad Alhaj; Rajkumar, S. Vincent; Dispenzieri, Angela; Gertz, Morie A.; Lacy, Martha Q.; Buadi, Francis K.; Hwa, Yi Lisa; Dingli, David; Kapoor, Prashant; Hayman, Suzanne R.; Lust, John A.; Kyle, Robert A.; Kumar, Shaji K.

    2015-01-01

    Normalization of the serum free light chain ratio (FLCr) with the absence of bone marrow monoclonal plasma cells following achievement of a complete response (CR) to therapy denotes a stringent CR in multiple myeloma (MM), and is associated with improved overall survival (OS). However, its value in patients achieving

  18. Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back.

    PubMed

    Alamo, Lorenzo; Li, Xiaochuan Edward; Espinoza-Fonseca, L Michel; Pinto, Antonio; Thomas, David D; Lehman, William; Padrón, Raúl

    2015-08-01

    Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence length analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation. PMID:26038302

  19. Microtubule-associated protein light chain 3 is involved in melanogenesis via regulation of MITF expression in melanocytes

    PubMed Central

    Yun, Woo Jin; Kim, Eun-Young; Park, Ji-Eun; Jo, Soo Youn; Bang, Seung Hyun; Chang, Eun-Ju; Chang, Sung Eun

    2016-01-01

    Although autophagy plays a role in melanogenesis by regulating melanosome degradation and biogenesis in melanocytes, a detailed understanding of the regulatory functions of autophagy factors is lacking. Here, we report a mechanistic link between microtubule-associated protein light chain 3 (LC3) activation and melanogenesis. We observed high expression of LC3 in melanosome-associated pigment-rich melanocytic nevi of sun-exposed skin, as indicated by patterns of melanosomal protein MART1 expression. Rapamycin-induced autophagy significantly increased the melanin index, tyrosinase activity and expression of several proteins linked to melanosome biogenesis, including microphthalmia transcription factor (MITF), pre-melanosome protein and tyrosinase, in Melan-a melanocytes. siRNA-mediated knockdown of LC3, but not beclin-1 or ATG5, decreased melanin content and tyrosinase activity. LC3 knockdown also markedly inhibited MITF expression and subsequent rapamycin-induced melanosome formation. More importantly, LC3 knockdown suppressed α-MSH-mediated melanogenesis by attenuating cAMP response element-binding protein (CREB) phosphorylation and MITF expression in Melan-a cells via decreased extracellular signal-regulated kinase (ERK) activity. Overexpression of constitutively active ERK reversed the effect of LC3 knockdown on CREB phosphorylation and MITF expression. These findings demonstrate that LC3 contributes to melanogenesis by increasing ERK-dependent MITF expression, thereby providing a mechanistic insight into the signaling network that links autophagy to melanogenesis. PMID:26814135

  20. Axonemal dynein light chain-1 locates at the microtubule-binding domain of the γ heavy chain

    PubMed Central

    Ichikawa, Muneyoshi; Saito, Kei; Yanagisawa, Haru-aki; Yagi, Toshiki; Kamiya, Ritsu; Yamaguchi, Shin; Yajima, Junichiro; Kushida, Yasuharu; Nakano, Kentaro; Numata, Osamu; Toyoshima, Yoko Y.

    2015-01-01

    The outer arm dynein (OAD) complex is the main propulsive force generator for ciliary/flagellar beating. In Chlamydomonas and Tetrahymena, the OAD complex comprises three heavy chains (α, β, and γ HCs) and >10 smaller subunits. Dynein light chain-1 (LC1) is an essential component of OAD. It is known to associate with the Chlamydomonas γ head domain, but its precise localization within the γ head and regulatory mechanism of the OAD complex remain unclear. Here Ni-NTA-nanogold labeling electron microscopy localized LC1 to the stalk tip of the γ head. Single-particle analysis detected an additional structure, most likely corresponding to LC1, near the microtubule-binding domain (MTBD), located at the stalk tip. Pull-down assays confirmed that LC1 bound specifically to the γ MTBD region. Together with observations that LC1 decreased the affinity of the γ MTBD for microtubules, we present a new model in which LC1 regulates OAD activity by modulating γ MTBD's affinity for the doublet microtubule. PMID:26399296

  1. Neurofilament Light Chain in Blood and CSF as Marker of Disease Progression in Mouse Models and in Neurodegenerative Diseases.

    PubMed

    Bacioglu, Mehtap; Maia, Luis F; Preische, Oliver; Schelle, Juliane; Apel, Anja; Kaeser, Stephan A; Schweighauser, Manuel; Eninger, Timo; Lambert, Marius; Pilotto, Andrea; Shimshek, Derya R; Neumann, Ulf; Kahle, Philipp J; Staufenbiel, Matthias; Neumann, Manuela; Maetzler, Walter; Kuhle, Jens; Jucker, Mathias

    2016-07-01

    A majority of current disease-modifying therapeutic approaches for age-related neurodegenerative diseases target their characteristic proteopathic lesions (α-synuclein, Tau, Aβ). To monitor such treatments, fluid biomarkers reflecting the underlying disease process are crucial. We found robust increases of neurofilament light chain (NfL) in CSF and blood in murine models of α-synucleinopathies, tauopathy, and β-amyloidosis. Blood and CSF NfL levels were strongly correlated, and NfL increases coincided with the onset and progression of the corresponding proteopathic lesions in brain. Experimental induction of α-synuclein lesions increased CSF and blood NfL levels, while blocking Aβ lesions attenuated the NfL increase. Consistently, we also found NfL increases in CSF and blood of human α-synucleinopathies, tauopathies, and Alzheimer's disease. Our results suggest that CSF and particularly blood NfL can serve as a reliable and easily accessible biomarker to monitor disease progression and treatment response in mouse models and potentially in human proteopathic neurodegenerative diseases.

  2. A Chlamydomonas Homologue of the Putative Murine t Complex Distorter Tctex-2 Is an Outer Arm Dynein Light Chain

    PubMed Central

    Patel-King, Ramila S.; Benashski, Sharon E.; Harrison, Alistair; King, Stephen M.

    1997-01-01

    Molecular analysis of a 19,000-Mr protein from the Chlamydomonas flagellum reveals that it is homologous to the t complex–encoded protein Tctex-2, which is a candidate for one of the distorter products that cause the extreme transmission ratio distortion (meiotic drive) of the murine t complex. The 19,000-Mr protein is extracted from the axoneme with 0.6 M NaCl and comigrates with the outer dynein arm in sucrose density gradients. This protein also is specifically missing in axonemes prepared from a mutant that does not assemble the outer arm. These data raise the possibility that Tctex-2 is a sperm flagellar dynein component. Combined with the recent identification of Tctex-1 (another distorter candidate) as a light chain of cytoplasmic dynein, these results lead to a biochemical model for how differential defects in spermiogenesis that result in the phenomenon of meiotic drive might be generated in wild-type vs t-bearing sperm. PMID:9166408

  3. Dynein light chain binding to a 3′-untranslated sequence mediates parathyroid hormone mRNA association with microtubules

    PubMed Central

    Epstein, Eyal; Sela-Brown, Alin; Ringel, Israel; Kilav, Rachel; King, Stephen M.; Benashski, Sharon E.; Yisraeli, Joel K.; Silver, Justin; Naveh-Many, Tally

    2000-01-01

    The 3′-untranslated region (UTR) of mRNAs binds proteins that determine mRNA stability and localization. The 3′-UTR of parathyroid hormone (PTH) mRNA specifically binds cytoplasmic proteins. We screened an expression library for proteins that bind the PTH mRNA 3′-UTR, and the sequence of 1 clone was identical to that of the dynein light chain LC8, a component of the dynein complexes that translocate cytoplasmic components along microtubules. Recombinant LC8 binds PTH mRNA 3′-UTR, as shown by RNA electrophoretic mobility shift assay. We showed that PTH mRNA colocalizes with microtubules in the parathyroid gland, as well as with a purified microtubule preparation from calf brain, and that this association was mediated by LC8. To our knowledge, this is the first report of a dynein complex protein binding an mRNA. The dynein complex may be the motor that is responsible for transporting mRNAs to specific locations in the cytoplasm and for the consequent is asymmetric distribution of translated proteins in the cell. PMID:10683380

  4. Diphosphorylated but not monophosphorylated myosin II regulatory light chain localizes to the midzone without its heavy chain during cytokinesis.

    PubMed

    Kondo, Tomo; Isoda, Rieko; Uchimura, Takashi; Sugiyama, Mutsumi; Hamao, Kozue; Hosoya, Hiroshi

    2012-01-13

    Myosin II is activated by the monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). Its ATPase activity is further enhanced by MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC). As these phosphorylated MRLCs are colocalized with their heavy chains at the contractile ring in dividing cells, we believe that the phosphorylated MRLC acts as a subunit of the activated myosin II during cytokinesis. However, the distinct role(s) of 1P- and 2P-MRLC during cytokinesis has not been elucidated. In this study, a monoclonal antibody (4F12) specific for 2P-MRLC was raised and used to examine the roles of 2P-MRLC in cultured mammalian cells. Our confocal microscopic observations using 4F12 revealed that 2P-MRLC localized to the contractile ring, and, unexpectedly, to the midzone also. Interestingly, 2P-MRLC did not colocalize with 1P-MRLC, myosin II heavy chain, and F-actin at the midzone. These results suggest that 2P-MRLC has a role different from that of 1P-MRLC at the midzone, and is not a subunit of myosin II. PMID:22166199

  5. The role of the N-terminus of the myosin essential light chain in cardiac muscle contraction

    PubMed Central

    Kazmierczak, Katarzyna; Xu, Yuanyuan; Jones, Michelle; Guzman, Georgianna; Hernandez, Olga M.; Kerrick, W. Glenn L.; Szczesna-Cordary, Danuta

    2011-01-01

    Summary To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43 amino acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of non-pathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin. PMID:19361417

  6. A mutation in the atrial-specific myosin light chain gene (MYL4) causes familial atrial fibrillation

    PubMed Central

    Orr, Nathan; Arnaout, Rima; Gula, Lorne J.; Spears, Danna A.; Leong-Sit, Peter; Li, Qiuju; Tarhuni, Wadea; Reischauer, Sven; Chauhan, Vijay S.; Borkovich, Matthew; Uppal, Shaheen; Adler, Arnon; Coughlin, Shaun R.; Stainier, Didier Y. R.; Gollob, Michael H.

    2016-01-01

    Atrial fibrillation (AF), the most common arrhythmia, is a growing epidemic with substantial morbidity and economic burden. Mechanisms underlying vulnerability to AF remain poorly understood, which contributes to the current lack of highly effective therapies. Recognizing mechanistic subtypes of AF may guide an individualized approach to patient management. Here, we describe a family with a previously unreported syndrome characterized by early-onset AF (age <35 years), conduction disease and signs of a primary atrial myopathy. Phenotypic penetrance was complete in all mutation carriers, although complete disease expressivity appears to be age-dependent. We show that this syndrome is caused by a novel, heterozygous p.Glu11Lys mutation in the atrial-specific myosin light chain gene MYL4. In zebrafish, mutant MYL4 leads to disruption of sarcomeric structure, atrial enlargement and electrical abnormalities associated with human AF. These findings describe the cause of a rare subtype of AF due to a primary, atrial-specific sarcomeric defect. PMID:27066836

  7. Interactions of Yeast Dynein with Dynein Light Chain and Dynactin: GENERAL IMPLICATIONS FOR INTRINSICALLY DISORDERED DUPLEX SCAFFOLDS IN MULTIPROTEIN ASSEMBLIES.

    PubMed

    Jie, Jing; Löhr, Frank; Barbar, Elisar

    2015-09-25

    Intrinsically disordered protein (IDP) duplexes composed of two IDP chains cross-linked by bivalent partner proteins form scaffolds for assembly of multiprotein complexes. The N-terminal domain of dynein intermediate chain (N-IC) is one such IDP that forms a bivalent scaffold with multiple dynein light chains including LC8, a hub protein that promotes duplex formation of diverse IDP partners. N-IC also binds a subunit of the dynein regulator, dynactin. Here we characterize interactions of a yeast ortholog of N-IC (N-Pac11) with yeast LC8 (Dyn2) or with the intermediate chain-binding subunit of yeast dynactin (Nip100). Residue level changes in Pac11 structure are monitored by NMR spectroscopy, and binding energetics are monitored by isothermal titration calorimetry (ITC). N-Pac11 is monomeric and primarily disordered except for a single α-helix (SAH) at the N terminus and a short nascent helix, LH, flanked by the two Dyn2 recognition motifs. Upon binding Dyn2, the only Pac11 residues making direct protein-protein interactions are in and immediately flanking the recognition motifs. Dyn2 binding also orders LH residues of Pac11. Upon binding Nip100, only Pac11 SAH residues make direct protein-protein interactions, but LH residues at a distant sequence position and L1 residues in an adjacent linker are also ordered. The long distance, ligand-dependent ordering of residues reveals new elements of dynamic structure within IDP linker regions.

  8. Chlamydia trachomatis inclusion membrane protein CT850 interacts with the dynein light chain DYNLT1 (Tctex1).

    PubMed

    Mital, Jeffrey; Lutter, Erika I; Barger, Alexandra C; Dooley, Cheryl A; Hackstadt, Ted

    2015-06-26

    Chlamydia trachomatis actively subverts the minus-end directed microtubule motor, dynein, to traffic along microtubule tracks to the Microtubule Organizing Center (MTOC) where it remains within a membrane bound replicative vacuole for the duration of its intracellular development. Unlike most substrates of the dynein motor, disruption of the dynactin cargo-linking complex by over-expression of the p50 dynamitin subunit does not inhibit C. trachomatis transport. A requirement for chlamydial protein synthesis to initiate this process suggests that a chlamydial product supersedes a requirement for p50 dynamitin. A yeast 2-hybrid system was used to screen the chlamydia inclusion membrane protein CT850 against a HeLa cell cDNA library and identified an interaction with the dynein light chain DYNLT1 (Tctex1). This interaction was at least partially dependent upon an (R/K-R/K-X-X-R/K) motif that is characteristic of DYNLT1 binding domains. CT850 expressed ectopically in HeLa cells localized at the MTOC and this localization is similarly dependent upon the predicted DYNLT1 binding domain. Furthermore, DYNLT1 is enriched at focal concentrations of CT850 on the chlamydial inclusion membrane that are known to interact with dynein and microtubules. Depletion of DYNLT1 disrupts the characteristic association of the inclusion membrane with centrosomes. Collectively, the results suggest that CT850 interacts with DYNLT1 to promote appropriate positioning of the inclusion at the MTOC.

  9. Slow motility in hair cells of the frog amphibian papilla: Myosin light chain-mediated shape change

    PubMed Central

    Farahbakhsh, Nasser A.; Narins, Peter M.

    2008-01-01

    Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca2+/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were recorded and compared. These cells respond to ionomycin with a tri-phasic shape change: an initial phase of iso-volumetric length decrease; a period of concurrent shortening and swelling; and the final phase of increase in both length and volume. We found that both the myosin light chain kinase inhibitor, ML-7, and antagonists of the multifunctional Ca2+/CaM-dependent kinases, KN-62 and KN-93, inhibit the iso-volumetric shortening phase of the response to ionomycin. The type 1 protein phosphatase inhibitors, calyculin A and okadaic acid induce minor shortening on their own, but do not significantly alter the phase 1 response. However, they appear to counter effects of the inhibitors of Ca2+/CaM-dependent kinases. We hypothesize that an active actomyosin-based process mediates the iso-volumetric shortening in the frog rostral amphibian papillar hair cells. PMID:18534795

  10. Slow motility in hair cells of the frog amphibian papilla: myosin light chain-mediated shape change.

    PubMed

    Farahbakhsh, Nasser A; Narins, Peter M

    2008-07-01

    Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca(2+)/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were recorded and compared. These cells respond to ionomycin with a tri-phasic shape change: an initial phase of iso-volumetric length decrease; a period of concurrent shortening and swelling; and the final phase of increase in both length and volume. We found that both the myosin light chain kinase inhibitor, ML-7, and antagonists of the multifunctional Ca(2+)/CaM-dependent kinases, KN-62 and KN-93, inhibit the iso-volumetric shortening phase of the response to ionomycin. The type 1 protein phosphatase inhibitors, calyculin A and okadaic acid induce minor shortening on their own, but do not significantly alter phase 1 response. However, they appear to counter effects of the inhibitors of Ca(2+)/CaM-dependent kinases. We hypothesize that an active actomyosin-based process mediates the iso-volumetric shortening in the frog rostral amphibian papillar hair cells.

  11. Resonance Assignments and Secondary Structure Analysis of Dynein Light Chain 8 by Magic Angle Spinning NMR Spectroscopy

    PubMed Central

    Sun, Shangjin; Butterworth, Andrew H.; Paramasivam, Sivakumar; Yan, Si; Lightcap, Christine M.; Williams, John C.; Polenova, Tatyana

    2012-01-01

    Dynein light chain LC8 is the smallest subunit of the dynein motor complex and has been shown to play important roles in both dynein dependent and dynein independent physiological functions via its interaction with a number of its binding partners. It has also been linked to pathogenesis including roles in viral infections and tumorigenesis. Structural information for LC8-target proteins is critical to understanding the underlying function of LC8 in these complexes. However, some LC8-target interactions are not amenable for structural characterization by conventional structural biology techniques due to their large size, low solubility and crystallization difficulties. Here, we report magic angle spinning (MAS) NMR studies of the homodimeric apo-LC8 protein as a first effort in addressing more complex, multi-partner LC8-based protein assemblies. We have established site-specific backbone and side chain resonance assignments for the majority of the residues of LC8, and show TALOS+ predicted torsion angles ϕ and ψ in close agreement with most residues in the published LC8 crystal structure. Data obtained through these studies will provide the first step toward using MAS NMR to examine the LC8 structure, which will eventually be used to investigate protein-protein interactions in larger systems, which cannot be determined by conventional structural studies. PMID:23243318

  12. Direct reduction of antigen receptor expression in polyclonal B cell populations developing in vivo results in light chain receptor editing.

    PubMed

    Shen, Shixue; Manser, Tim

    2012-01-01

    Secondary Ab V region gene segment rearrangement, termed receptor editing, is a major mechanism contributing to B lymphocyte self-tolerance. However, the parameters that determine whether a B cell undergoes editing are a current subject of debate. We tested the role that the level of BCR expression plays in the regulation of receptor editing in a polyclonal population of B cells differentiating in vivo. Expression of a short hairpin RNA for κ L chain RNA in B cells resulted in reduction in levels of this RNA and surface BCRs. Strikingly, fully mature and functional B cells that developed in vivo and efficiently expressed the short hairpin RNA predominantly expressed BCRs containing λ light chains. This shift in L chain repertoire was accompanied by inhibition of development, increased Rag gene expression, and increased λ V gene segment-cleavage events at the immature B cell stage. These data demonstrated that reducing the translation of BCRs that are members of the natural repertoire at the immature B cell stage is sufficient to promote editing.

  13. Dynein Light Chain 1 Regulates Dynamin-mediated F-Actin Assembly during Sperm Individualization in DrosophilaD⃞

    PubMed Central

    Ghosh-Roy, Anindya; Desai, Bela S.; Ray, Krishanu

    2005-01-01

    Toward the end of spermiogenesis, spermatid nuclei are compacted and the clonally related spermatids individualize to become mature and active sperm. Studies in Drosophila showed that caudal end-directed movement of a microfilament-rich structure, called investment cone, expels the cytoplasmic contents of individual spermatids. F-actin dynamics plays an important role in this process. Here we report that the dynein light chain 1 (DLC1) of Drosophila is involved in two separate cellular processes during sperm individualization. It is enriched around spermatid nuclei during postelongation stages and plays an important role in the dynein-dynactin–dependent rostral retention of the nuclei during this period. In addition, DDLC1 colocalizes with dynamin along investment cones and regulates F-actin assembly at this organelle by retaining dynamin along the cones. Interestingly, we found that this process does not require the other subunits of cytoplasmic dynein-dynactin complex. Altogether, these observations suggest that DLC1 could independently regulate multiple cellular functions and established a novel role of this protein in F-actin assembly in Drosophila. PMID:15829565

  14. Structures of Clostridium Botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility

    SciTech Connect

    Silvaggi,N.; Boldt, G.; Hixon, M.; Kennedy, J.; Tzipori, S.; Janda, K.; Allen, K.

    2007-01-01

    The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1' site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC.

  15. A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin.

    PubMed

    Facemyer, K C; Cremo, C R

    1992-01-01

    We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.

  16. OMEGA: The Open MEG Archive.

    PubMed

    Niso, Guiomar; Rogers, Christine; Moreau, Jeremy T; Chen, Li-Yuan; Madjar, Cecile; Das, Samir; Bock, Elizabeth; Tadel, François; Evans, Alan C; Jolicoeur, Pierre; Baillet, Sylvain

    2016-01-01

    In contrast with other imaging modalities, there is presently a scarcity of fully open resources in magnetoencephalography (MEG) available to the neuroimaging community. Here we present a collaborative effort led by the McConnell Brain Imaging Centre of the Montreal Neurological Institute, and the Université de Montréal to build and share a centralised repository to curate MEG data in raw and processed form for open dissemination. The Open MEG Archive (OMEGA, omega.bic.mni.mcgill.ca) is bound to become a continuously expanding repository of multimodal data with a primary focus on MEG, in addition to storing anatomical MRI volumes, demographic participant data and questionnaires, and other forms of electrophysiological data such as EEG. The OMEGA initiative offers both the technological framework for multi-site MEG data aggregation, and serves as one of the largest freely available resting-state and eventually task-related MEG datasets presently available.

  17. Serum Free Light Chains

    MedlinePlus

    ... changes in the ratio of kappa and lambda production, which indicate an excess of one clone of ... test to detect abnormal monoclonal protein (M-protein) production and to calculate a kappa/lambda free light ...

  18. Examination of Glycan Profiles from IgG-Depleted Human Immunoglobulins Facilitated by Microscale Affinity Chromatography

    PubMed Central

    Svoboda, Martin; Mann, Benjamin F.; Goetz, John A.; Novotny, Milos V.

    2012-01-01

    Among the most important proteins involved in the disease and healing processes are the immunoglobulins (Igs). Although many of the Igs have been studied through proteomics, aside from IgG, immunoglobulin carbohydrates have not been extensively characterized in different states of health. It seems valuable to develop techniques that permit us to understand changes in the structures and abundances of Ig glycans in the context of disease onset and progression. We have devised a strategy for characterization of the glycans for the Ig classes other than IgG (i.e. A, D, E, and M) that contain kappa light chains, while using only a few microliters of biological material. First, we designed a microcolumn containing the recombinant Protein L that was immobilized on macroporous silica particles. A similarly designed Protein G microcolumn was utilized to first perform an on-line depletion of the IgG from the sample, human blood serum, and thereby facilitate enrichment of the other Igs. While only 3 μL of serum were used in these analyses, we were able to recover a significantly-enriched fraction of non-IgG immunoglobulins. The enrichment properties of the Protein L column were characterized using a highly sensitive label-free quantitative proteomics LC-MS/MS approach, and the glycomic profiles of enriched immunoglobulins were measured by MALDI-TOF-MS. As a proof-of-principle, a comparative study was conducted using blood serum from a small group of lung cancer patients and a group of age-matched cancer-free individuals to demonstrate that the method is suitable for investigation of glycosylation changes in disease. The results were in agreement with a glycomic investigation of whole blood serum from a much larger lung cancer cohort. PMID:22360417

  19. Humoral immunoglobulins of the white sturgeon, Acipenser transmontanus: partial characterization of and recognition with monoclonal antibodies.

    PubMed

    Adkison, M A; Basurco, B; Hedrick, R P

    1996-01-01

    White sturgeon (Acipenser transmontanus) immunoglobulin (Ig) was purified from serum by two methods, ion-exchange chromatography and gel filtration and precipitation of the euglobulin fraction. The purity of these immunoglobulin preparations was confirmed by gel electrophoresis. Sequence analysis of the N-terminal amino acids confirmed that the purified protein was immunoglobulin. The major portion of the immunoglobulin preparation consisted of two proteins with estimated molecular weights (m.w.) of 870 and 170 kDa. The m.w. of the H- and L-chains of the purified Ig were 73 and 27-30 kDa, respectively, as determined by SDS-PAGE. Ion-exchange purified Ig was used to immunize mice for the production of monoclonal antibodies. This resulted in the production of six stable hybrids that recognized sturgeon Ig, two specific for heavy chain and four specific for light chain. The two anti-H-chain mabs were highly specific for white sturgeon Ig while all four anti-L-chain mabs cross reacted with Ig from green sturgeon (A. medirostris), Atlantic sturgeon (A. oxyrhynchus oxyrhynchus), shovelnose sturgeon (Scaphirhynchus platorynchus), and paddlefish (Polyodon spathula), (all Chondrosteans), but not with channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss) or striped bass (Morone saxatilis). The mabs were used to enumerate the percentage of sIg+ lymphocytes in the peripheral blood of white sturgeon by flow cytometry. The percentage of cells positively stained with the mabs ranged from 12 to 28%. In a comparison of mabs with polyclonal rabbit anti-sturgeon Ig serum by ELISA the mabs produced a larger signal and less background than the polyclonal serum.

  20. An Increase in the Omega-6/Omega-3 Fatty Acid Ratio Increases the Risk for Obesity

    PubMed Central

    Simopoulos, Artemis P.

    2016-01-01

    In the past three decades, total fat and saturated fat intake as a percentage of total calories has continuously decreased in Western diets, while the intake of omega-6 fatty acid increased and the omega-3 fatty acid decreased, resulting in a large increase in the omega-6/omega-3 ratio from 1:1 during evolution to 20:1 today or even higher. This change in the composition of fatty acids parallels a significant increase in the prevalence of overweight and obesity. Experimental studies have suggested that omega-6 and omega-3 fatty acids elicit divergent effects on body fat gain through mechanisms of adipogenesis, browning of adipose tissue, lipid homeostasis, brain-gut-adipose tissue axis, and most importantly systemic inflammation. Prospective studies clearly show an increase in the risk of obesity as the level of omega-6 fatty acids and the omega-6/omega-3 ratio increase in red blood cell (RBC) membrane phospholipids, whereas high omega-3 RBC membrane phospholipids decrease the risk of obesity. Recent studies in humans show that in addition to absolute amounts of omega-6 and omega-3 fatty acid intake, the omega-6/omega-3 ratio plays an important role in increasing the development of obesity via both AA eicosanoid metabolites and hyperactivity of the cannabinoid system, which can be reversed with increased intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). A balanced omega-6/omega-3 ratio is important for health and in the prevention and management of obesity. PMID:26950145

  1. An Increase in the Omega-6/Omega-3 Fatty Acid Ratio Increases the Risk for Obesity.

    PubMed

    Simopoulos, Artemis P

    2016-03-02

    In the past three decades, total fat and saturated fat intake as a percentage of total calories has continuously decreased in Western diets, while the intake of omega-6 fatty acid increased and the omega-3 fatty acid decreased, resulting in a large increase in the omega-6/omega-3 ratio from 1:1 during evolution to 20:1 today or even higher. This change in the composition of fatty acids parallels a significant increase in the prevalence of overweight and obesity. Experimental studies have suggested that omega-6 and omega-3 fatty acids elicit divergent effects on body fat gain through mechanisms of adipogenesis, browning of adipose tissue, lipid homeostasis, brain-gut-adipose tissue axis, and most importantly systemic inflammation. Prospective studies clearly show an increase in the risk of obesity as the level of omega-6 fatty acids and the omega-6/omega-3 ratio increase in red blood cell (RBC) membrane phospholipids, whereas high omega-3 RBC membrane phospholipids decrease the risk of obesity. Recent studies in humans show that in addition to absolute amounts of omega-6 and omega-3 fatty acid intake, the omega-6/omega-3 ratio plays an important role in increasing the development of obesity via both AA eicosanoid metabolites and hyperactivity of the cannabinoid system, which can be reversed with increased intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). A balanced omega-6/omega-3 ratio is important for health and in the prevention and management of obesity.

  2. Observation of an excited charm baryon Omega c* decaying to Omega c0gamma.

    PubMed

    Aubert, B; Bona, M; Boutigny, D; Couderc, F; Karyotakis, Y; Lees, J P; Poireau, V; Tisserand, V; Zghiche, A; Grauges, E; Palano, A; Chen, J C; Qi, N D; Rong, G; Wang, P; Zhu, Y S; Eigen, G; Ofte, I; Stugu, B; Abrams, G S; Battaglia, M; Brown, D N; Button-Shafer, J; Cahn, R N; Charles, E; Gill, M S; Groysman, Y; Jacobsen, R G; Kadyk, J A; Kerth, L T; Kolomensky, Yu G; Kukartsev, G; Lynch, G; Mir, L M; Orimoto, T J; Pripstein, M; Roe, N A; Ronan, M T; Wenzel, W A; del Amo Sanchez, P; Barrett, M; Ford, K E; Hart, A J; Harrison, T J; Hawkes, C M; Watson, A T; Held, T; Koch, H; Lewandowski, B; Pelizaeus, M; Peters, K; Schroeder, T; Steinke, M; Boyd, J T; Burke, J P; Cottingham, W N; Walker, D; Asgeirsson, D J; Cuhadar-Donszelmann, T; Fulsom, B G; Hearty, C; Knecht, N S; Mattison, T S; McKenna, J A; Khan, A; Kyberd, P; Saleem, M; Sherwood, D J; Teodorescu, L; Blinov, V E; Bukin, A D; Druzhinin, V P; Golubev, V B; Onuchin, A P; Serednyakov, S I; Skovpen, Yu I; Solodov, E P; Todyshev, K Yu; Bondioli, M; Bruinsma, M; Chao, M; Curry, S; Eschrich, I; Kirkby, D; Lankford, A J; Lund, P; Mandelkern, M; Mommsen, R K; Roethel, W; Stoker, D P; Abachi, S; Buchanan, C; Foulkes, S D; Gary, J W; Long, O; Shen, B C; Wang, K; Zhang, L; Hadavand, H K; Hill, E J; Paar, H P; Rahatlou, S; Sharma, V; Berryhill, J W; Campagnari, C; Cunha, A; Dahmes, B; Hong, T M; Kovalskyi, D; Richman, J D; Beck, T W; Eisner, A M; Flacco, C J; Heusch, C A; Kroseberg, J; Lockman, W S; Nesom, G; Schalk, T; Schumm, B A; Seiden, A; Spradlin, P; Williams, D C; Wilson, M G; Albert, J; Chen, E; Dvoretskii, A; Fang, F; Hitlin, D G; Narsky, I; Piatenko, T; Porter, F C; Ryd, A; Mancinelli, G; Meadows, B T; Mishra, K; Sokoloff, M D; Blanc, F; Bloom, P C; Chen, S; Ford, W T; Hirschauer, J F; Kreisel, A; Nagel, M; Nauenberg, U; Olivas, A; Ruddick, W O; Smith, J G; Ulmer, K A; Wagner, S R; Zhang, J; Chen, A; Eckhart, E A; Soffer, A; Toki, W H; Wilson, R J; Winklmeier, F; Zeng, Q; Altenburg, D D; Feltresi, E; Hauke, A; Jasper, H; Merkel, J; Petzold, A; Spaan, B; Brandt, T; Klose, V; Lacker, H M; Mader, W F; Nogowski, R; Schubert, J; Schubert, K R; Schwierz, R; Sundermann, J E; Volk, A; Bernard, D; Bonneaud, G R; Latour, E; Thiebaux, Ch; Verderi, M; Clark, P J; Gradl, W; Muheim, F; Playfer, S; Robertson, A I; Xie, Y; Andreotti, M; Bettoni, D; Bozzi, C; Calabrese, R; Cibinetto, G; Luppi, E; Negrini, M; Petrella, A; Piemontese, L; Prencipe, E; Anulli, F; Baldini-Ferroli, R; Calcaterra, A; de Sangro, R; Finocchiaro, G; Pacetti, S; Patteri, P; Peruzzi, I M; Piccolo, M; Rama, M; Zallo, A; Buzzo, A; Contri, R; Lo Vetere, M; Macri, M M; Monge, M R; Passaggio, S; Patrignani, C; Robutti, E; Santroni, A; Tosi, S; Brandenburg, G; Chaisanguanthum, K S; Morii, M; Wu, J; Dubitzky, R S; Marks, J; Schenk, S; Uwer, U; Bhimji, W; Bowerman, D A; Dauncey, P D; Egede, U; Flack, R L; Nash, J A; Nikolich, M B; Panduro Vazquez, W; Bard, D J; Behera, P K; Chai, X; Charles, M J; Mallik, U; Meyer, N T; Ziegler, V; Cochran, J; Crawley, H B; Dong, L; Eyges, V; Meyer, W T; Prell, S; Rosenberg, E I; Rubin, A E; Gritsan, A V; Denig, A G; Fritsch, M; Schott, G; Arnaud, N; Davier, M; Grosdidier, G; Höcker, A; Le Diberder, F; Lepeltier, V; Lutz, A M; Oyanguren, A; Pruvot, S; Rodier, S; Roudeau, P; Schune, M H; Stocchi, A; Wang, W F; Wormser, G; Cheng, C H; Lange, D J; Wright, D M; Chavez, C A; Forster, I J; Fry, J R; Gabathuler, E; Gamet, R; George, K A; Hutchcroft, D E; Payne, D J; Schofield, K C; Touramanis, C; Bevan, A J; Di Lodovico, F; Menges, W; Sacco, R; Cowan, G; Flaecher, H U; Hopkins, D A; Jackson, P S; McMahon, T R; Ricciardi, S; Salvatore, F; Wren, A C; Brown, D N; Davis, C L; Allison, J; Barlow, N R; Barlow, R J; Chia, Y M; Edgar, C L; Lafferty, G D; Naisbit, M T; Williams, J C; Yi, J I; Chen, C; Hulsbergen, W D; Jawahery, A; Lae, C K; Roberts, D A; Simi, G; Blaylock, G; Dallapiccola, C; Hertzbach, S S; Li, X; Moore, T B; Saremi, S; Staengle, H; Cowan, R; Sciolla, G; Sekula, S J; Spitznagel, M; Taylor, F; Yamamoto, R K; Kim, H; Mclachlin, S E; Patel, P M; Robertson, S H; Lazzaro, A; Lombardo, V; Palombo, F; Bauer, J M; Cremaldi, L; Eschenburg, V; Godang, R; Kroeger, R; Sanders, D A; Summers, D J; Zhao, H W; Brunet, S; Côté, D; Simard, M; Taras, P; Viaud, F B; Nicholson, H; Cavallo, N; De Nardo, G; Fabozzi, F; Gatto, C; Lista, L; Monorchio, D; Paolucci, P; Piccolo, D; Sciacca, C; Baak, M A; Raven, G; Snoek, H L; Jessop, C P; LoSecco, J M; Allmendinger, T; Benelli, G; Corwin, L A; Gan, K K; Honscheid, K; Hufnagel, D; Jackson, P D; Kagan, H; Kass, R; Rahimi, A M; Regensburger, J J; Ter-Antonyan, R; Wong, Q K; Blount, N L; Brau, J; Frey, R; Igonkina, O; Kolb, J A; Lu, M; Rahmat, R; Sinev, N B; Strom, D; Strube, J; Torrence, E; Gaz, A; Margoni, M; Morandin, M; Pompili, A; Posocco, M; Rotondo, M; Simonetto, F; Stroili, R; Voci, C; Benayoun, M; Briand, H; Chauveau, J; David, P; Del Buono, L; de la Vaissière, Ch; Hamon, O; Hartfiel, B L; Leruste, Ph; Malclès, J; Ocariz, J; Roos, L; Therin, G; Gladney, L; Biasini, M; Covarelli, R; Angelini, C; Batignani, G; Bettarini, S; Bucci, F; Calderini, G; Carpinelli, M; Cenci, R; Forti, F; Giorgi, M A; Lusiani, A; Marchiori, G; Mazur, M A; Morganti, M; Neri, N; Paoloni, E; Rizzo, G; Walsh, J J; Haire, M; Judd, D; Wagoner, D E; Biesiada, J; Danielson, N; Elmer, P; Lau, Y P; Lu, C; Olsen, J; Smith, A J S; Telnov, A V; Bellini, F; Cavoto, G; D'Orazio, A; del Re, D; Di Marco, E; Faccini, R; Ferrarotto, F; Ferroni, F; Gaspero, M; Li Gioi, L; Mazzoni, M A; Morganti, S; Piredda, G; Polci, F; Tehrani, F Safai; Voena, C; Ebert, M; Schröder, H; Waldi, R; Adye, T; De Groot, N; Franek, B; Olaiya, E O; Wilson, F F; Aleksan, R; Emery, S; Gaidot, A; Ganzhur, S F; Hamel de Monchenault, G; Kozanecki, W; Legendre, M; Vasseur, G; Yèche, Ch; Zito, M; Chen, X R; Liu, H; Park, W; Purohit, M V; Wilson, J R; Allen, M T; Aston, D; Bartoldus, R; Bechtle, P; Berger, N; Claus, R; Coleman, J P; Convery, M R; Cristinziani, M; Dingfelder, J C; Dorfan, J; Dubois-Felsmann, G P; Dujmic, D; Dunwoodie, W; Field, R C; Glanzman, T; Gowdy, S J; Graham, M T; Grenier, P; Halyo, V; Hast, C; Hryn'ova, T; Innes, W R; Kelsey, M H; Kim, P; Leith, D W G S; Li, S; Luitz, S; Luth, V; Lynch, H L; MacFarlane, D B; Marsiske, H; Messner, R; Muller, D R; O'Grady, C P; Ozcan, V E; Perazzo, A; Perl, M; Pulliam, T; Ratcliff, B N; Roodman, A; Salnikov, A A; Schindler, R H; Schwiening, J; Snyder, A; Stelzer, J; Su, D; Sullivan, M K; Suzuki, K; Swain, S K; Thompson, J M; Va'vra, J; van Bakel, N; Weaver, M; Weinstein, A J R; Wisniewski, W J; Wittgen, M; Wright, D H; Yarritu, A K; Yi, K; Young, C C; Burchat, P R; Edwards, A J; Majewski, S A; Petersen, B A; Roat, C; Wilden, L; Ahmed, S; Alam, M S; Bula, R; Ernst, J A; Jain, V; Pan, B; Saeed, M A; Wappler, F R; Zain, S B; Bugg, W; Krishnamurthy, M; Spanier, S M; Eckmann, R; Ritchie, J L; Satpathy, A; Schilling, C J; Schwitters, R F; Izen, J M; Lou, X C; Ye, S; Bianchi, F; Gallo, F; Gamba, D; Bomben, M; Bosisio, L; Cartaro, C; Cossutti, F; Della Ricca, G; Dittongo, S; Lanceri, L; Vitale, L; Azzolini, V; Lopez-March, N; Martinez-Vidal, F; Banerjee, Sw; Bhuyan, B; Brown, C M; Fortin, D; Hamano, K; Kowalewski, R; Nugent, I M; Roney, J M; Sobie, R J; Back, J J; Harrison, P F; Latham, T E; Mohanty, G B; Pappagallo, M; Band, H R; Chen, X; Cheng, B; Dasu, S; Datta, M; Flood, K T; Hollar, J J; Kutter, P E; Mellado, B; Mihalyi, A; Pan, Y; Pierini, M; Prepost, R; Wu, S L; Yu, Z; Neal, H

    2006-12-01

    We report the first observation of an excited singly charmed baryon Omega c* (css) in the radiative decay Omega c0gamma, where the Omega c0 baryon is reconstructed in the decays to the final states Omega(-)pi+, Omega(-)pi+pi0, Omega(-)pi+pi(-)pi+, and Xi(-)K(-)pi+pi+. This analysis is performed using a data set of 230.7 fb(-1) collected by the BABAR detector at the PEP-II asymmetric-energy B factory at the Stanford Linear Accelerator Center. The mass difference between the Omega c* and the Omega c0 baryons is measured to be 70.8+/-1.0(stat)+/-1.1(syst) MeV/c2. We also measure the ratio of inclusive production cross sections of Omega c* and Omega c0 in e+e(-) annihilation. PMID:17280195

  3. Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier.

    PubMed

    Ashokkumar, Balasubramaniem; Nabokina, Svetlana M; Ma, Thomas Y; Said, Hamid M

    2009-09-01

    The reduced folate carrier (RFC) is a major folate transport system in mammalian cells. RFC is highly expressed in the intestine and believed to play a role in folate absorption. Studies from our laboratory and others have characterized different aspects of the intestinal folate absorption process, but little is known about possible existence of accessory protein(s) that interacts with RFC and influences its physiology and/or cell biology. We investigated this issue by employing a bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library using the large intracellular loop between transmembrane domains 6 and 7 of the human RFC (hRFC) as bait. Our screening has resulted in the identification of dynein light chain road block-1 (DYNLRB1) as an interacting partner with hRFC. Existence of a direct protein-protein interaction between hRFC and DYNLRB1 was confirmed by in vitro pull-down assay and in vivo mammalian two-hybrid luciferase assay and coimmunoprecipitation analysis. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells demonstrated colocalization of DYNLRB1 with hRFC. Coexpression of DYNLRB1 with hRFC led to a significant (P < 0.05) increase in folate uptake. On the other hand, inhibiting the endogenous DYNLRB1 with gene-specific small interfering RNA or pharmacologically with a specific inhibitor (vanadate) led to a significant (P < 0.05) decrease in folate uptake. This study demonstrates for the first time the identification of DYNLRB1 as an interacting protein partner with hRFC. Furthermore, DYNLRB1 appears to influence the function and cell biology of hRFC.

  4. Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle.

    PubMed

    Hong, Feng; Brizendine, Richard K; Carter, Michael S; Alcala, Diego B; Brown, Avery E; Chattin, Amy M; Haldeman, Brian D; Walsh, Michael P; Facemyer, Kevin C; Baker, Josh E; Cremo, Christine R

    2015-10-01

    Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot-labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto-myosin and MLCK-myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting "stuck" on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle.

  5. The essential light chain N-terminal extension alters force and fiber kinetics in mouse cardiac muscle.

    PubMed

    Miller, Mark S; Palmer, Bradley M; Ruch, Stuart; Martin, Lisa A; Farman, Gerrie P; Wang, Yuan; Robbins, Jeffrey; Irving, Thomas C; Maughan, David W

    2005-10-14

    The functional significance of the actin-binding region at the N terminus of the cardiac myosin essential light chain (ELC) remains elusive. In a previous experiment, the endogenous ventricular ELC was replaced with a protein containing a 10-amino acid deletion at positions 5-14 (ELC1vDelta5-14, referred to as 1vDelta5-14), a region that interacts with actin. 1vDelta5-14 mice showed no discernable mutant phenotype in skinned ventricular strips. However, because the myofilament lattice swells upon skinning, the mutant phenotype may have been concealed by the inability of the ELC to reach the actin-binding site. Using the same mouse model, we repeated earlier measurements and performed additional experiments on skinned strips osmotically compressed to the intact lattice spacing as determined by x-ray diffraction. 1vDelta5-14 mice exhibited decreased maximum isometric tension without a change in calcium sensitivity. The decreased force was most evident in 5-6-month-old mice compared with 13-15-month-old mice and may account for the greater ventricular wall thickness in young 1vDelta5-14 mice compared with age-matched controls. No differences were observed in unloaded shortening velocity at maximum calcium activation. However, 1vDelta5-14 mice exhibited a significant difference in the frequency at which minimum complex modulus amplitude occurred, indicating a change in cross-bridge kinetics. We hypothesize that the ELC N-terminal extension interaction with actin inhibits the reversal of the power stroke, thereby increasing isometric force. Our results strongly suggest that an interaction between residues 5-14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance.

  6. A shrimp pacifastin light chain-like inhibitor: molecular identification and role in the control of the prophenoloxidase system.

    PubMed

    Sangsuriya, Pakkakul; Charoensapsri, Walaiporn; Chomwong, Sudarat; Senapin, Saengchan; Tassanakajon, Anchalee; Amparyup, Piti

    2016-01-01

    Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp.

  7. Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle

    PubMed Central

    Hong, Feng; Brizendine, Richard K.; Carter, Michael S.; Alcala, Diego B.; Brown, Avery E.; Chattin, Amy M.; Haldeman, Brian D.; Walsh, Michael P.; Facemyer, Kevin C.; Baker, Josh E.

    2015-01-01

    Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot–labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto–myosin and MLCK–myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting “stuck” on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle. PMID:26415568

  8. Phosphorylated Myosin Light Chain 2 (p-MLC2) as a Molecular Marker of Antemortem Coronary Artery Spasm

    PubMed Central

    Li, Liliang; Li, Yuhua; Lin, Junyi; Jiang, Jieqing; He, Meng; Sun, Daming; Zhao, Ziqin; Shen, Yiwen; Xue, Aimin

    2016-01-01

    Background It is not uncommon that only mild coronary artery stenosis is grossly revealed after a system autopsy. While coronary artery spasm (CAS) is the suspected mechanism of these deaths, no specific biomarker has been identified to suggest antemortem CAS. Material/Methods To evaluate the potential of using phosphorylated myosin light chain 2 (p-MLC2) as a diagnostic marker of antemortem CAS, human vascular smooth muscle cells (VSMCs) were cultured and treated with common vasoconstrictors, including prostaglandins F2α (PGF2α), acetylcholine (ACh), and 5-hydroxy tryptamine (5-HT). The p-MLC2 level was examined in the cultured cells using Western blot analysis and in a rat model of spasm provocation tests using immunohistochemistry (IHC). Effects of increased p-MLC2 level on VSMCs contractile activities were assessed in vitro using confocal immunofluorescence assay. Four fatal cases with known antemortem CAS were collected and subject to p-MLC2 detection. Results The p-MLC2 was significantly increased in VSMCs after treatments with vasoconstrictors and in the spasm provocation tests. Myofilament was well-organized and densely stained in VSMCs with high p-MLC2 level, but disarrayed in VSMCs with low p-MLC2 level. Three of the 4 autopsied cases showed strongly positive staining of p-MLC2 at the stenosed coronary segment and the adjacent interstitial small arteries. The fourth case was autopsied at the 6th day after death and showed negative-to-mild positive staining of p-MLC2. Conclusions p-MLC2 might be a useful marker for diagnosis of antemortem CAS. Autopsy should be performed as soon as possible to collect coronary arteries for detection of p-MLC2. PMID:27643564

  9. Interleukin-18 facilitates neutrophil transmigration via myosin light chain kinase-dependent disruption of occludin, without altering epithelial permeability.

    PubMed

    Lapointe, Tamia K; Buret, Andre G

    2012-02-01

    Compromised epithelial barrier function and tight junction alterations are hallmarks of a number of gastrointestinal disorders, including inflammatory bowel disease (IBD). Increased levels of IL-18 have been observed in mucosal samples from Crohn's disease and ulcerative colitis patients. Remarkably, several reports have demonstrated that immunological or genetic blockage of IL-18 ameliorates the severity of colitis in multiple in vivo models of IBD. Nevertheless, the effects of IL-18 on intestinal epithelial barrier function remain unclear. We hypothesized that IL-18 could disrupt intestinal epithelial barrier structure and function, thus contributing to tissue damage in the context of IBD. The aims of the present study were to determine the effects of IL-18 on epithelial barrier structure and function and to characterize the mechanisms involved in these modulatory properties. Human colonic epithelial Caco-2 monolayers were coincubated with IL-18 for 24 h and processed for immunocytochemistry, immunoblotting, quantitative PCR, and permeability measurements (transepithelial resistance, FITC-dextran fluxes, and bacterial translocation). Our findings indicate that IL-18 selectively disrupts tight junctional occludin, without affecting the distribution pattern of claudin-4, claudin-5, zonula occludens-1, or E-cadherin. This effect coincided with a significant increase in myosin light chain kinase (MLCK) protein levels and activity. Pharmacological inhibition of MLCK and NF-κB prevented IL-18-induced loss of occludin. Although too subtle to alter paracellular permeability, these fine changes correlated with an MLCK-dependent increase in neutrophil transepithelial migration. In conclusion, our data suggest that IL-18 may potentiate inflammation in the context of IBD by facilitating neutrophil transepithelial migration via MLCK-dependent disruption of tight junctional occludin.

  10. Non–Muscle Myosin Light Chain Kinase Isoform Is a Viable Molecular Target in Acute Inflammatory Lung Injury

    PubMed Central

    Mirzapoiazova, Tamara; Moitra, Jaideep; Moreno-Vinasco, Liliana; Sammani, Saad; Turner, Jerry R.; Chiang, Eddie T.; Evenoski, Carrie; Wang, Ting; Singleton, Patrick A.; Huang, Yong; Lussier, Yves A.; Watterson, D. Martin; Dudek, Steven M.; Garcia, Joe G. N.

    2011-01-01

    Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non–muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (∼50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody–conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (∼70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (∼40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6–signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation. PMID:20139351

  11. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments.

    PubMed

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2016-05-24

    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease.

  12. Molecular cloning, characterisation and mRNA expression analysis of the sheep myosin light chain 1 gene.

    PubMed

    Zhang, Chunlan; Wang, Guizhi; Ji, Zhibin; Liu, Zhaohua; Hou, Lei; Liu, Guanqing; Wang, Jianmin

    2015-09-10

    The complete cDNA sequence of the sheep MYL1 (Myosin light chain 1) gene was cloned using RT-PCR, 5' RACE and 3' RACE. We obtained two alternatively spliced isoforms of the MYL1 gene, MYL1a and MYL1b, which are 849 and 1046bp in length and encode proteins composed of 150 and 192 amino acid residues, respectively. And the GenBank accession numbers of MYL1a and MYL1b full-length cDNA sequences that we cloned are KJ700419 and KJ710701, respectively. Neither protein was predicted to have a signal peptide, but both were predicted to have several N-glycosylation and phosphorylation sites. More than half of the secondary structure of these proteins was predicted to be α-helical. The human MYL2 protein (1m8q.1.C) is the most similar in tertiary structure. Sequence alignment showed that the sheep MYL1a protein shares more than 92% amino acid sequence similar with Mus musculus, Homo sapiens, Rattus norvegicus, Sus scrofa and Gallus gallus and that the MYL1b protein shares more than 93% amino acid sequence similar with M. musculus, H. sapiens, R. norvegicus, Bos taurus and Oryctolagus cuniculus. Transcription profile analyses of various tissues indicated that the sheep MYL1a and MYL1b mRNAs were highly but differentially expressed in the longissimus dorsi. Moreover, the expression levels of these genes in the longissimus dorsi differed between Dorper and Small-tailed Han sheep. These results serve as a foundation for further investigations of the function of the sheep MYL1 gene. PMID:25911560

  13. Phosphorylated Myosin Light Chain 2 (p-MLC2) as a Molecular Marker of Antemortem Coronary Artery Spasm.

    PubMed

    Li, Liliang; Li, Yuhua; Lin, Junyi; Jiang, Jieqing; He, Meng; Sun, Daming; Zhao, Ziqin; Shen, Yiwen; Xue, Aimin

    2016-01-01

    BACKGROUND It is not uncommon that only mild coronary artery stenosis is grossly revealed after a system autopsy. While coronary artery spasm (CAS) is the suspected mechanism of these deaths, no specific biomarker has been identified to suggest antemortem CAS. MATERIAL AND METHODS To evaluate the potential of using phosphorylated myosin light chain 2 (p-MLC2) as a diagnostic marker of antemortem CAS, human vascular smooth muscle cells (VSMCs) were cultured and treated with common vasoconstrictors, including prostaglandins F2α (PGF2α), acetylcholine (ACh), and 5-hydroxy tryptamine (5-HT). The p-MLC2 level was examined in the cultured cells using Western blot analysis and in a rat model of spasm provocation tests using immunohistochemistry (IHC). Effects of increased p-MLC2 level on VSMCs contractile activities were assessed in vitro using confocal immunofluorescence assay. Four fatal cases with known antemortem CAS were collected and subject to p-MLC2 detection. RESULTS The p-MLC2 was significantly increased in VSMCs after treatments with vasoconstrictors and in the spasm provocation tests. Myofilament was well-organized and densely stained in VSMCs with high p-MLC2 level, but disarrayed in VSMCs with low p-MLC2 level. Three of the 4 autopsied cases showed strongly positive staining of p-MLC2 at the stenosed coronary segment and the adjacent interstitial small arteries. The fourth case was autopsied at the 6th day after death and showed negative-to-mild positive staining of p-MLC2. CONCLUSIONS p-MLC2 might be a useful marker for diagnosis of antemortem CAS. Autopsy should be performed as soon as possible to collect coronary arteries for detection of p-MLC2. PMID:27643564

  14. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

    PubMed Central

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2016-01-01

    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease. PMID:27162358

  15. The LC7 Light Chains of Chlamydomonas Flagellar Dyneins Interact with Components Required for Both Motor Assembly and Regulation

    PubMed Central

    DiBella, Linda M.; Sakato, Miho; Patel-King, Ramila S.; Pazour, Gregory J.; King, Stephen M.

    2004-01-01

    Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced ∼20% in axonemes isolated from strains lacking inner arm I1 and are ∼80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles ∼30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm γ heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins. PMID:15304520

  16. Berberine ameliorates severe acute pancreatitis‑induced intestinal barrier dysfunction via a myosin light chain phosphorylation‑dependent pathway.

    PubMed

    Liang, Hong-Yin; Chen, Tao; Yan, Hong-Tao; Huang, Zhu; Tang, Li-Jun

    2014-05-01

    Berberine is a traditional drug used to treat gastrointestinal disorders in China and has been demonstrated to attenuate intestinal barrier dysfunction in certain animal models. However, the effects of berberine on pancreatitis-induced intestinal barrier dysfunction are yet to be fully elucidated. This study aimed to investigate the effect of berberine pretreatment on the attenuation of intestinal barrier dysfunction induced by severe acute pancreatitis (SAP). A total of 36 rats were randomly divided into Sham, SAP and SAP plus berberine groups. Pancreatitis was induced using retrograde injection of 3% Na-taurocholate into the pancreatic duct. Histological examinations of the pancreas were performed and intestinal barrier dysfunction was characterized by histological measurements and the assessment of serum diamine oxidase activity and endotoxin levels. Zonula occludens-1 and occludin mRNA and protein expression, as well as myosin light chain (MLC) phosphorylation, were assessed. SAP rat models were successfully established. Berberine treatment was found to have no significant effect on the histological changes in the pancreas, but was observed to ameliorate the intestinal mucosal barrier damage and membrane permeability associated with SAP. Although berberine exerted minimal effects on tight junction proteins in the ilea of SAP rats, it was observed to significantly inhibit SAP-induced MLC phosphorylation. To the best of our knowledge, this is the first study to demonstrate that berberine attenuates SAP‑induced intestinal barrier dysfunction in vivo. In addition, this study shows that the effect of berberine on intestinal barrier function may be associated with the inhibition of SAP‑induced upregulation of MLC phosphorylation.

  17. Kinesin Light Chain 1 Suppression Impairs Human Embryonic Stem Cell Neural Differentiation and Amyloid Precursor Protein Metabolism

    PubMed Central

    Killian, Rhiannon L.; Flippin, Jessica D.; Herrera, Cheryl M.; Almenar-Queralt, Angels; Goldstein, Lawrence S. B.

    2012-01-01

    The etiology of sporadic Alzheimer disease (AD) is largely unknown, although evidence implicates the pathological hallmark molecules amyloid beta (Aβ) and phosphorylated Tau. Work in animal models suggests that altered axonal transport caused by Kinesin-1 dysfunction perturbs levels of both Aβ and phosphorylated Tau in neural tissues, but the relevance of Kinesin-1 dependent functions to the human disease is unknown. To begin to address this issue, we generated human embryonic stem cells (hESC) expressing reduced levels of the kinesin light chain 1 (KLC1) Kinesin-1 subunit to use as a source of human neural cultures. Despite reduction of KLC1, undifferentiated hESC exhibited apparently normal colony morphology and pluripotency marker expression. Differentiated neural cultures derived from KLC1-suppressed hESC contained neural rosettes but further differentiation revealed obvious morphological changes along with reduced levels of microtubule-associated neural proteins, including Tau and less secreted Aβ, supporting the previously established connection between KLC1, Tau and Aβ. Intriguingly, KLC1-suppressed neural precursors (NPs), isolated using a cell surface marker signature known to identify cells that give rise to neurons and glia, unlike control cells, failed to proliferate. We suggest that KLC1 is required for normal human neural differentiation, ensuring proper metabolism of AD-associated molecules APP and Tau and for proliferation of NPs. Because impaired APP metabolism is linked to AD, this human cell culture model system will not only be a useful tool for understanding the role of KLC1 in regulating the production, transport and turnover of APP and Tau in neurons, but also in defining the essential function(s) of KLC1 in NPs and their progeny. This knowledge should have important implications for human neurodevelopmental and neurodegenerative diseases. PMID:22272245

  18. Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

    PubMed

    Belvitch, Patrick; Adyshev, Djanybek; Elangovan, Venkateswaran R; Brown, Mary E; Naureckas, Caitlin; Rizzo, Alicia N; Siegler, Jessica H; Garcia, Joe G N; Dudek, Steven M

    2014-09-01

    Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function. PMID:25072537

  19. Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

    SciTech Connect

    Jin, R.; Sikorra, S.; Stegmann, C.M.; Pich, A.; Binz, T.; Brunger, A.T.

    2009-06-01

    Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote a-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the a-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.

  20. Recombinant enterokinase light chain with affinity tag: expression from Saccharomyces cerevisiae and its utilities in fusion protein technology.

    PubMed

    Choi, S I; Song, H W; Moon, J W; Seong, B L

    2001-12-20

    Enterokinase and recombinant enterokinase light chain (rEK(L)) have been used widely to cleave fusion proteins with the target sequence of (Asp)(4)-Lys. In this work, we show that their utility as a site-specific cleavage agent is compromised by sporadic cleavage at other sites, albeit at low levels. Further degradation of the fusion protein in cleavage reaction is due to an intrinsic broad specificity of the enzyme rather than to the presence of contaminating proteases. To offer facilitated purification from fermentation broth and efficient removal of rEK(L) after cleavage reaction, thus minimizing unwanted cleavage of target protein, histidine affinity tag was introduced into rEK(L). Utilizing the secretion enhancer peptide derived from the human interleukin 1 beta, the recombinant EK(L) was expressed in Saccharomyces cerevisiae and efficiently secreted into culture medium. The C-terminal His-tagged EK(L) was purified in a single-step procedure on nickel affinity chromatography. It retained full enzymatic activity similar to that of EK(L), whereas the N-terminal His-tagged EK(L) was neither efficiently purified nor had any enzymatic activity. After cleavage reaction of fusion protein, the C-terminal His-tagged EK(L) was efficiently removed from the reaction mixture by a single passage through nickel-NTA spin column. The simple affinity tag renders rEK(L) extremely useful for purification, post-cleavage removal, recovery, and recycling and will broaden the utility and the versatility of the enterokinase for the production of recombinant proteins. PMID:11745150

  1. Kinetic and Motor Functions Mediated by Distinct Regions of the Regulatory Light Chain of Smooth Muscle Myosin1,2

    PubMed Central

    Ni, Shaowei; Hong, Feng; Brewer, Paul D.; Ikebe, Mitsuo; Onishi, Hirofumi; Baker, Jonathan E.; Facemyer, Kevin C.; Cremo, Christine R.

    2009-01-01

    To understand the importance of selected regions of the regulatory light chain (RLC) for phosphorylation-dependent regulation of smooth muscle myosin (SMM), we expressed three heavy meromyosins (HMMs) containing the following RLC mutants; K12E in a critical region of the phosphorylation domain, GTDP95-98/AAAA in the central hinge, and R160C a putative binding residue for phosphorylated S19. Single-turnover actin-activated Mg2+-ATPase (Vmax and Katpase) and in vitro actin sliding velocities were examined for both unphosphorylated (up-) and phosphorylated (p-) states. Turnover rates for the upstate (0.007-0.030 s-1) and velocities (no motion) for all constructs were not significantly different from the up-wild type (WT) indicating that they were completely turned off. The apparent binding constants for actin in the presence of ATP (Katpase) were too weak to measure as expected for fully regulated constructs. For p-HMM containing GTDP/AAAA, we found that both ATPase and motility were normal. The data suggest that the native sequence in the central hinge between the two lobes of the RLC is not required for turning the HMM off and on both kinetically and mechanically. For p-HMM containing R160C, all parameters were normal, suggesting that R160C is not involved in coordination of the phosphorylated S19. For p-HMM containing K12E, the Vmax was 64% and actin sliding velocity was ∼50% of WT, suggesting that K12 is an important residue for the ability to sense or to promote the conformational changes required for kinetic and mechanical activation. PMID:19635597

  2. The nondigestible disaccharide epilactose increases paracellular Ca absorption via rho-associated kinase- and myosin light chain kinase-dependent mechanisms in rat small intestines.

    PubMed

    Suzuki, Takuya; Nishimukai, Megumi; Takechi, Maki; Taguchi, Hidenori; Hamada, Shigeki; Yokota, Atsushi; Ito, Susumu; Hara, Hiroshi; Matsui, Hirokazu

    2010-02-10

    We previously showed that epilactose, a nondigestible disaccharide, increased calcium (Ca) absorption in the small intestines of rats. Here, we explored the mechanism(s) underlying the epilactose-mediated promotion of Ca absorption in a ligated intestinal segment of anesthetized rats. The addition of epilactose to the luminal solution increased Ca absorption and chromium (Cr)-EDTA permeability, a paracellular indicator, with a strong correlation (R = 0.93) between these changes. Epilactose induced the phosphorylation of myosin regulatory light chains (MLCs), which is known to activate the paracellular route, without any change in the association of tight junction proteins with the actin cytoskeleton. The epilactose-mediated promotion of the Ca absorption was suppressed by specific inhibitors of myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). These results indicate that epilactose increases paracellular Ca absorption in the small intestine of rats through the induction of MLC phosphorylation via MLCK- and ROCK-dependent mechanisms.

  3. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle.

    PubMed

    Poetter, K; Jiang, H; Hassanzadeh, S; Master, S R; Chang, A; Dalakas, M C; Rayment, I; Sellers, J R; Fananapazir, L; Epstein, N D

    1996-05-01

    The muscle myosins and hexomeric proteins consisting of two heavy chains and two pairs of light chains, the latter called essential (ELC) and regulatory (RLC). The light chains stabilize the long alpha helical neck of the myosin head. Their function in striated muscle, however, is only partially understood. We report here the identification of distinct missense mutations in a skeletal/ventricular ELC and RLC, each of which are associated with a rare variant of cardiac hypertrophy as well as abnormal skeletal muscle. We show that myosin containing the mutant ELC has abnormal function, map the mutant residues on the three-dimensional structure of myosin and suggest that the mutations disrupt the stretch activation response of the cardiac papillary muscles.

  4. Measurement of the Omega0(c) lifetime

    SciTech Connect

    Iori, M.; Ayan, A.S.; Akgun, U.; Alkhazov, G.; Amaro-Reyes, J.; Atamantchouk, A.G.; Balatz, M.Y.; Blanco-Covarrubias, A.; Bondar, N.F.; Cooper, P.S.; Dauwe, L.J.; /Ball State U. /Bogazici U. /Carnegie Mellon U. /Rio de Janeiro, CBPF /Fermilab /Serpukhov, IHEP /Beijing, Inst. High Energy Phys. /Moscow, ITEP /Heidelberg, Max Planck Inst. /Moscow State U. /St. Petersburg, INP

    2007-01-01

    The authors report a precise measurement of the {Omega}{sub c}{sup 0} lifetime. The data were taken by the SELEX (E781) experiment using 600 GeV/c {Sigma}{sup -}, {pi}{sup -} and p beams. The measurement has been made using 83 {+-} 19 reconstructed {Omega}{sub c}{sup 0} in the {Omega}{sup -} {pi}{sup -}{pi}{sup +}{pi}{sup +} and {Omega}{sup -} {pi}{sup +} decay modes. The lifetime of the {Omega}{sub c}{sup 0} is measured to be 65 {+-} 13(stat) {+-} 9(sys) fs.

  5. Confocal Cornea Microscopy Detects Involvement of Corneal Nerve Fibers in a Patient with Light-Chain Amyloid Neuropathy Caused by Multiple Myeloma: A Case Report

    PubMed Central

    Sturm, Dietrich; Schmidt-Wilcke, Tobias; Greiner, Tineke; Maier, Christoph; Schargus, Marc; Tegenthoff, Martin; Vorgerd, Matthias

    2016-01-01

    Changes in the subbasal corneal plexus detected by confocal cornea microscopy (CCM) have been described for various types of neuropathy. An involvement of these nerves within light-chain (AL) amyloid neuropathy (a rare cause of polyneuropathy) has never been shown. Here, we report on a case of a patient suffering from neuropathy caused by AL amyloidosis and underlying multiple myeloma. Small-fiber damage was detected by CCM. PMID:27482195

  6. A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.

    PubMed

    Bookwalter, Carol S; Kelsen, Anne; Leung, Jacqueline M; Ward, Gary E; Trybus, Kathleen M

    2014-10-31

    Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.

  7. Dynamin-1-like protein (Dnm1L) interaction with kinesin light chain 1 (KLC1) through the tetratricopeptide repeat (TPR) domains.

    PubMed

    Jang, Won Hee; Jeong, Young Joo; Choi, Sun Hee; Kim, Sang-Jin; Urm, Sang-Hwa; Seog, Dae-Hyun

    2014-01-01

    Kinesin light chain 1 (KLC1) mediates binding of KIF5 motor to specific cargo. Using the yeast two-hybrid screening, we found that mitochondrial fission protein dynamin-1-like protein (Dnm1L) interacted with KLC1, but not KIF5. Dnm1L and KLC1 were co-localized in cultured cells. These results suggest that KLC1 may play a potential role in post-fission mitochondrial transport.

  8. Role of kinesin light chain-2 of kinesin-1 in the traffic of Na,K-ATPase-containing vesicles in alveolar epithelial cells

    PubMed Central

    Trejo, Humberto E.; Lecuona, Emilia; Grillo, Doris; Szleifer, Igal; Nekrasova, Oksana E.; Gelfand, Vladimir I.; Sznajder, Jacob I.

    2010-01-01

    Recruitment of the Na,K-ATPase to the plasma membrane of alveolar epithelial cells results in increased active Na+ transport and fluid clearance in a process that requires an intact microtubule network. However, the microtubule motors involved in this process have not been identified. In the present report, we studied the role of kinesin-1, a plus-end microtubule molecular motor that has been implicated in the movement of organelles in the Na,K-ATPase traffic. We determined by confocal microscopy and biochemical assays that kinesin-1 and the Na,K-ATPase are present in the same membranous cellular compartment. Knockdown of kinesin-1 heavy chain (KHC) or the light chain-2 (KLC2), but not of the light chain-1 (KLC1), decreased the movement of Na,K-ATPase-containing vesicles when compared to sham siRNA-transfected cells (control group). Thus, a specific isoform of kinesin-1 is required for microtubule-dependent recruitment of Na,K-ATPase to the plasma membrane, which is of physiological significance—Trejo, H. E., Lecuona, E., Grillo, D., Szleifer, I., Nekrasova, O. E., Gelfand, V. I., Sznajder, J. I. Role of kinesin light chain-2 of kinesin-1 in the traffic of Na,K-ATPase-containing vesicles in alveolar epithelial cells. PMID:19773350

  9. The novel zinc finger protein dASCIZ regulates mitosis in Drosophila via an essential role in dynein light-chain expression.

    PubMed

    Zaytseva, Olga; Tenis, Nora; Mitchell, Naomi; Kanno, Shin-ichiro; Yasui, Akira; Heierhorst, Jörg; Quinn, Leonie M

    2014-02-01

    The essential zinc finger protein ASCIZ (also known as ATMIN, ZNF822) plays critical roles during lung organogenesis and B cell development in mice, where it regulates the expression of dynein light chain (DYNLL1/LC8), but its functions in other species including invertebrates are largely unknown. Here we report the identification of the Drosophila ortholog of ASCIZ (dASCIZ) and show that loss of dASCIZ function leads to pronounced mitotic delays with centrosome and spindle positioning defects during development, reminiscent of impaired dynein motor functions. Interestingly, similar mitotic and developmental defects were observed upon knockdown of the DYNLL/LC8-type dynein light chain Cutup (Ctp), and dASCIZ loss-of-function phenotypes could be suppressed by ectopic Ctp expression. Consistent with a genetic function of dASCIZ upstream of Ctp, we show that loss of dASCIZ led to reduced endogenous Ctp mRNA and protein levels and dramatically reduced Ctp-LacZ reporter gene activity in vivo, indicating that dASCIZ regulates development and mitosis as a Ctp transcription factor. We speculate that the more severe mitotic defects in the absence of ASCIZ in flies compared to mice may be due to redundancy with a second, ASCIZ-independent, Dynll2 gene in mammals in contrast to a single Ctp gene in Drosophila. Altogether, our data demonstrate that ASCIZ is an evolutionary highly conserved transcriptional regulator of dynein light-chain levels and a novel regulator of mitosis in flies.

  10. Genetic analysis of dilated cardiomyopathy--HLA and immunoglobulin genes may confer susceptibility.

    PubMed

    Nishi, H; Kimura, A; Fukuta, S; Kusukawa, R; Kawamura, K; Nimura, Y; Nagano, M; Yasuda, H; Kawai, C; Sugimoto, T

    1992-10-01

    To identify genetic factors in the immune system which control the susceptibility to dilated cardiomyopathy (DCM), HLA class II DNA typing was performed in 61 Japanese patients, using PCR/SSO probe analyses. The frequencies of HLA-DQB1*0503 (15% vs 5%; RR = 3.06, chi 2 = 7.19) and DQB1*0604 (21% vs 10%; RR = 2.41, chi 2 = 6.20) were significantly increased and that of HLA-DQB1*0502 (RR = 1.74) was slightly increased in the DCM patients. The frequency of DQB1*0303 (16% vs 31%; RR = 0.44, chi 2 = 5.16) was significantly decreased in the patients. The increased HLA-DQB1 alleles have a histidine residue in common at the 30th codon for the HLA-DQ beta chain. Among the genetic markers studied by Southern blot analyses, IGLV (immunoglobulin lambda light chain, pV3.3) showed a strong association with DCM, i.e. A2/A2 genotype was found in 37.7% of patients whereas it was observed in only 18.9% of the control subjects (RR = 2.6, chi 2 = 7.77). The frequency of this genotype was higher in patients under age 45 years at the time of diagnosis (45.5%, RR = 3.6, chi 2 = 10.02). These results suggest that HLA and immunoglobulin genes are closely linked to susceptibility to DCM.

  11. A Heterologous Reporter Defines the Role of the Tetanus Toxin Interchain Disulfide in Light-Chain Translocation

    PubMed Central

    Zuverink, Madison; Chen, Chen; Przedpelski, Amanda; Blum, Faith C.

    2015-01-01

    Botulinum neurotoxins (BoNTs) and tetanus toxin (TeNT) are the most potent toxins for humans and elicit unique pathologies due to their ability to traffic within motor neurons. BoNTs act locally within motor neurons to elicit flaccid paralysis, while retrograde TeNT traffics to inhibitory neurons within the central nervous system (CNS) to elicit spastic paralysis. BoNT and TeNT are dichain proteins linked by an interchain disulfide bond comprised of an N-terminal catalytic light chain (LC) and a C-terminal heavy chain (HC) that encodes an LC translocation domain (HCT) and a receptor-binding domain (HCR). LC translocation is the least understood property of toxin action, but it involves low pH, proteolysis, and an intact interchain disulfide bridge. Recently, Pirazzini et al. (FEBS Lett 587:150–155, 2013, http://dx.doi.org/10.1016/j.febslet.2012.11.007) observed that inhibitors of thioredoxin reductase (TrxR) blocked TeNT and BoNT action in cerebellar granular neurons. In the current study, an atoxic TeNT LC translocation reporter was engineered by fusing β-lactamase to the N terminus of TeNT [βlac-TeNT(RY)] to investigate LC translocation in primary cortical neurons and Neuro-2a cells. βlac-TeNT(RY) retained the interchain disulfide bond, showed ganglioside-dependent binding to neurons, required acidification to promote βlac translocation, and was sensitive to auranofin, an inhibitor of thioredoxin reductase. Mutation of βlac-TeNT(RY) at C439S and C467S eliminated the interchain disulfide bond and inhibited βlac translocation. These data support the requirement of an intact interchain disulfide for LC translocation and imply that disulfide reduction is a prerequisite for LC delivery into the host cytosol. The data also support a model that LC translocation proceeds from the C to the N terminus. βlac-TeNT(RY) is the first reporter system to measure translocation by an AB single-chain toxin in intact cells. PMID:25895970

  12. Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40).

    PubMed

    Van Lierop, Jacquelyn E; Wilson, David P; Davis, Jonathan P; Tikunova, Svetlana; Sutherland, Cindy; Walsh, Michael P; Johnson, J David

    2002-02-22

    Calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays a key role in activation of smooth muscle contraction. A soybean isoform of CaM, SCaM-4 (77% identical to human CaM) fails to activate MLCK, whereas SCaM-1 (90.5% identical to human CaM) is as effective as CaM. We exploited this difference to gain insights into the structural requirements in CaM for activation of MLCK. A chimera (domain I of SCaM-4 and domains II-IV of SCaM-1) behaved like SCaM4, and analysis of site-specific mutants of SCaM-1 indicated that K30E and G40D mutations were responsible for the reduction in activation of MLCK. Competition experiments showed that SCaM-4 binds to the CaM-binding site of MLCK with high affinity. Replacement of CaM in skinned smooth muscle by exogenous CaM or SCaM-1, but not SCaM-4, restored Ca(2+)-dependent contraction. K30E/M36I/G40D SCaM-1 was a poor activator of contraction, but site-specific mutants, K30E, M36I and G40D, each restored Ca(2+)-induced contraction to CaM-depleted skinned smooth muscle, consistent with their capacity to activate MLCK. Interpretation of these results in light of the high-resolution structures of (Ca(2+))(4)-CaM, free and complexed with the CaM-binding domain of MLCK, indicates that a surface domain containing Lys(30) and Gly(40) and residues from the C-terminal domain is created upon binding to MLCK, formation of which is required for activation of MLCK. Interactions between this activation domain and a region of MLCK distinct from the known CaM-binding domain are required for removal of the autoinhibitory domain from the active site, i.e., activation of MLCK, or this domain may be required to stabilize the conformation of (Ca(2+))(4)-CaM necessary for MLCK activation.

  13. Rearrangement and expression of endogenous immunoglobulin genes occur in many murine B cells expressing transgenic membrane IgM.

    PubMed

    Stall, A M; Kroese, F G; Gadus, F T; Sieckmann, D G; Herzenberg, L A; Herzenberg, L A

    1988-05-01

    Transgenic mice carrying immunoglobulin genes coding for mu heavy chain and kappa light chain have been used to study the mechanisms involved in allelic and isotypic exclusion. We report here that individual cells from transgenic mice carrying a functionally rearranged mu heavy chain gene (capable of generating both membrane and secreted forms of IgM) can rearrange an endogenous mu heavy chain gene and simultaneously produce both transgenic and endogenous IgM. These "double-producing" cells express both endogenous and transgenic IgM in the cytoplasm (detected by immunohistology) and on the cell surface (detected by multiparameter fluorescence-activated cell sorter analysis). In addition, they secrete mixed IgM molecules containing both transgenic and endogenous mu heavy chains (detected in serum by radioimmune assay). The transgenic mice studied also have relatively large numbers of cells that produce endogenous immunoglobulin in the absence of detectable transgenic immunoglobulin ("endogenous-only cells"). The mechanisms that generate double-producing cells and endogenous-only cells appear to be under genetic control because the frequencies of these B-cell populations are characteristic for a given transgenic line. Thus, our findings indicate that more is involved in triggering allelic exclusion than the simple presence or absence of membrane mu heavy chains (as has been previously postulated).

  14. Murray and the Omega Minus

    SciTech Connect

    Samios, N.P.

    2010-08-20

    The exciting findings and activities in particle physics in the 50's and 60's will be discussed from an experimentalist's viewpoint. Particular emphasis will be placed on the description of several crucial discoveries (including the omega minus) and on the remarkable insight, guidance, and major contributions of Murray Gell-Mann to the understanding of the symmetry of hadrons which led to the development of the standard model of the strong interactions.

  15. Murray and the Omega Minus

    NASA Astrophysics Data System (ADS)

    Samios, Nicholas P.

    The exciting findings and activities in particle physics in the 50's and 60's will be discussed from an experimentalist's viewpoint. Particular emphasis will be placed on the description of several crucial discoveries (including the omega minus) and on the remarkable insight, guidance, and major contributions of Murray Gell-Mann to the understanding of the symmetry of hadrons which led to the development of the standard model of the strong interactions.

  16. Murray and the Omega Minus

    NASA Astrophysics Data System (ADS)

    Samios, Nicholas P.

    2011-11-01

    The exciting findings and activities in particle physics in the 50's and 60's will be discussed from an experimentalist's viewpoint. Particular emphasis will be placed on the description of several crucial discoveries (including the omega minus) and on the remarkable insight, guidance, and major contributions of Murray Gell-Mann to the understanding of the symmetry of hadrons which led to the development of the standard model of the strong interactions.

  17. Omega-X micromachining system

    DOEpatents

    Miller, Donald M.

    1978-01-01

    A micromachining tool system with X- and omega-axes is used to machine spherical, aspherical, and irregular surfaces with a maximum contour error of 100 nonometers (nm) and surface waviness of no more than 0.8 nm RMS. The omega axis, named for the angular measurement of the rotation of an eccentric mechanism supporting one end of a tool bar, enables the pulse increments of the tool toward the workpiece to be as little as 0 to 4.4 nm. A dedicated computer coordinates motion in the two axes to produce the workpiece contour. Inertia is reduced by reducing the mass pulsed toward the workpiece to about one-fifth of its former value. The tool system includes calibration instruments to calibrate the micromachining tool system. Backlash is reduced and flexing decreased by using a rotary table and servomotor to pulse the tool in the omega-axis instead of a ball screw mechanism. A thermally-stabilized spindle rotates the workpiece and is driven by a motor not mounted on the micromachining tool base through a torque-smoothing pulley and vibrationless rotary coupling. Abbe offset errors are almost eliminated by tool setting and calibration at spindle center height. Tool contour and workpiece contour are gaged on the machine; this enables the source of machining errors to be determined more readily, because the workpiece is gaged before its shape can be changed by removal from the machine.

  18. Omega-3 deficiency impairs honey bee learning.

    PubMed

    Arien, Yael; Dag, Arnon; Zarchin, Shlomi; Masci, Tania; Shafir, Sharoni

    2015-12-22

    Deficiency in essential omega-3 polyunsaturated fatty acids (PUFAs), particularly the long-chain form of docosahexaenoic acid (DHA), has been linked to health problems in mammals, including many mental disorders and reduced cognitive performance. Insects have very low long-chain PUFA concentrations, and the effect of omega-3 deficiency on cognition in insects has not been studied. We show a low omega-6:3 ratio of pollen collected by honey bee colonies in heterogenous landscapes and in many hand-collected pollens that we analyzed. We identified Eucalyptus as an important bee-forage plant particularly poor in omega-3 and high in the omega-6:3 ratio. We tested the effect of dietary omega-3 deficiency on olfactory and tactile associative learning of the economically highly valued honey bee. Bees fed either of two omega-3-poor diets, or Eucalyptus pollen, showed greatly reduced learning abilities in conditioned proboscis-extension assays compared with those fed omega-3-rich diets, or omega-3-rich pollen mixture. The effect on performance was not due to reduced sucrose sensitivity. Omega-3 deficiency also led to smaller hypopharyngeal glands. Bee brains contained high omega-3 concentrations, which were only slightly affected by diet, suggesting additional peripheral effects on learning. The shift from a low to high omega-6:3 ratio in the Western human diet is deemed a primary cause of many diseases and reduced mental health. A similar shift seems to be occurring in bee forage, possibly an important factor in colony declines. Our study shows the detrimental effect on cognitive performance of omega-3 deficiency in a nonmammal. PMID:26644556

  19. Omega-3 deficiency impairs honey bee learning

    PubMed Central

    Arien, Yael; Dag, Arnon; Zarchin, Shlomi; Masci, Tania

    2015-01-01

    Deficiency in essential omega-3 polyunsaturated fatty acids (PUFAs), particularly the long-chain form of docosahexaenoic acid (DHA), has been linked to health problems in mammals, including many mental disorders and reduced cognitive performance. Insects have very low long-chain PUFA concentrations, and the effect of omega-3 deficiency on cognition in insects has not been studied. We show a low omega-6:3 ratio of pollen collected by honey bee colonies in heterogenous landscapes and in many hand-collected pollens that we analyzed. We identified Eucalyptus as an important bee-forage plant particularly poor in omega-3 and high in the omega-6:3 ratio. We tested the effect of dietary omega-3 deficiency on olfactory and tactile associative learning of the economically highly valued honey bee. Bees fed either of two omega-3–poor diets, or Eucalyptus pollen, showed greatly reduced learning abilities in conditioned proboscis-extension assays compared with those fed omega-3–rich diets, or omega-3–rich pollen mixture. The effect on performance was not due to reduced sucrose sensitivity. Omega-3 deficiency also led to smaller hypopharyngeal glands. Bee brains contained high omega-3 concentrations, which were only slightly affected by diet, suggesting additional peripheral effects on learning. The shift from a low to high omega-6:3 ratio in the Western human diet is deemed a primary cause of many diseases and reduced mental health. A similar shift seems to be occurring in bee forage, possibly an important factor in colony declines. Our study shows the detrimental effect on cognitive performance of omega-3 deficiency in a nonmammal. PMID:26644556

  20. Women and omega-3 Fatty acids.

    PubMed

    Saldeen, Pia; Saldeen, Tom

    2004-10-01

    Omega-3 fatty acids (omega-3 FA) are constituents of the membranes of all cells in the body and are precursors of locally produced hormones, eicosanoids, which are important in the prevention and treatment of various diseases, especially in women. Omega-3 FA are of interest in some of the most common conditions affecting women. One mechanism underlying dysmenorrhea is a disturbed balance between antiinflammatory, vasodilator eicosanoids derived from omega-3 FA and proinflammatory, vasoconstrictor eicosanoids derived from omega-6 FA. Increased intake of omega-3 FA can reverse the symptoms in this condition by decreasing the amount of omega-6 FA in cell membranes. An increased prostacyclin/thromboxane ratio induced by omega-3 FA can facilitate pregnancy in women with infertility problems by increasing uterine blood flow. Supplementation with omega-3 FA during pregnancy lowers the risk of premature birth and can increase the length of pregnancy and birth weight by altering the balance of eicosanoids involved in labor and promote fetal growth by improving placental blood flow. Intake of omega-3 FA during pregnancy and breast feeding may facilitate the child's brain development. There is also some evidence that supplementation with omega-3 FA might help to prevent preeclampsia, postpartum depression, menopausal problems, postmenopausal osteoporosis, and breast cancer. Furthermore, because elevated triglyceride levels are associated with cardiovascular disease, especially in women; and because omega-3 FA have powerful effects on triglycerides, women in particular gain from an increased intake of these fatty acids. This is especially important in women receiving hormone therapy, which can increase triglyceride levels. The quality of the omega-3 FA preparation is important. It should have an appropriate antioxidant content not to induce lipid peroxidation, and its content of dioxin and polychlorinated biphenyls (PCBs) should be well below the established safe limit.

  1. Omega-3 deficiency impairs honey bee learning.

    PubMed

    Arien, Yael; Dag, Arnon; Zarchin, Shlomi; Masci, Tania; Shafir, Sharoni

    2015-12-22

    Deficiency in essential omega-3 polyunsaturated fatty acids (PUFAs), particularly the long-chain form of docosahexaenoic acid (DHA), has been linked to health problems in mammals, including many mental disorders and reduced cognitive performance. Insects have very low long-chain PUFA concentrations, and the effect of omega-3 deficiency on cognition in insects has not been studied. We show a low omega-6:3 ratio of pollen collected by honey bee colonies in heterogenous landscapes and in many hand-collected pollens that we analyzed. We identified Eucalyptus as an important bee-forage plant particularly poor in omega-3 and high in the omega-6:3 ratio. We tested the effect of dietary omega-3 deficiency on olfactory and tactile associative learning of the economically highly valued honey bee. Bees fed either of two omega-3-poor diets, or Eucalyptus pollen, showed greatly reduced learning abilities in conditioned proboscis-extension assays compared with those fed omega-3-rich diets, or omega-3-rich pollen mixture. The effect on performance was not due to reduced sucrose sensitivity. Omega-3 deficiency also led to smaller hypopharyngeal glands. Bee brains contained high omega-3 concentrations, which were only slightly affected by diet, suggesting additional peripheral effects on learning. The shift from a low to high omega-6:3 ratio in the Western human diet is deemed a primary cause of many diseases and reduced mental health. A similar shift seems to be occurring in bee forage, possibly an important factor in colony declines. Our study shows the detrimental effect on cognitive performance of omega-3 deficiency in a nonmammal.

  2. The Effect of Low Omega-3/Omega-6 Ratio on Auditory Nerve Conduction in Rat Pups.

    PubMed

    Farahani, Saeid; Motasaddi Zarandy, Masoud; Hassanzadeh, Gholamreza; Shidfar, Farzad; Jalaie, Shohreh; Rahimi, Vida

    2015-01-01

    The biological effects of omega-3 and omega-6 fatty acids are determined by their mutual interactions. This interaction extremely affects various functions. Lower consumption of omega-3 during gestation leads to various disorders, even in hearing. We aimed to assess the effect of low omega-3/omega-6 ratios on auditory nerve conduction. In this experimental study, the auditory brainstem response test was performed on 24-day-old rat (n=14). The rats were divided into case (low omega-3/omega-6 ratio during gestation and lactation) and control groups. Variables such as P1, P3, and P4 absolute latency period, interpeaks (P3-P4, P1-P3, and P1-P4), and P4/P1 amplitude ratio were measured. We found an increased P4 omega-3/omega-6 ratio in the group with a low omega-3/omega-6 ratio (P<0.01). No significant difference was observed in the P1 and P3 absolute latency period between the studied groups  (P>0.05).  Also, no significant difference was observed between the groups with respect to the P1-P3 interpeak latency (IPL) periods (P>0.05); while the P1-P4 and P3-P4 IPLs were significantly increased in the group with a low omega-3/omega-6 ratio (P<0.05). The P4/P1 amplitude ratio significantly decreased in the group with a low omega-3/omega-6 ratio (P<0.05). Results confirmed the negative effects of low omega-3/omega-6 ratio on the auditory system and hearing.

  3. Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains

    PubMed Central

    1992-01-01

    Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid

  4. Omega-3 Fatty Acids and Pregnancy

    PubMed Central

    Coletta, Jaclyn M; Bell, Stacey J; Roman, Ashley S

    2010-01-01

    Omega-3 fatty acids are essential fatty acids that must be consumed in the diet. Adequate consumption of omega-3 fatty acids is vitally important during pregnancy as they are critical building blocks of fetal brain and retina. Omega-3 fatty acids may also play a role in determining the length of gestation and in preventing perinatal depression. The most biologically active forms of omega-3 fatty acids are docosahexaenoic acid and eicosapentaenoic acid, which are primarily derived from marine sources such as seafood and algae. Recent surveys, however, indicate that pregnant women in the United States and in other countries eat little fish and therefore do not consume enough omega-3 fatty acids, primarily due to concern about the adverse effects of mercury and other contaminants on the developing fetus. This review discusses the benefits of omega-3 fatty acid consumption during pregnancy and provides guidelines for obstetricians advising patients. PMID:21364848

  5. FY15 LLNL OMEGA Experimental Programs

    SciTech Connect

    Heeter, R. F.; Baker, K. L.; Barrios, M. A.; Beckwith, M. A.; Casey, D. T.; Celliers, P. M.; Chen, H.; Coppari, F.; Fournier, K. B.; Fratanduono, D. E.; Frenje, J.; Huntington, C. M.; Kraus, R. G.; Lazicki, A. E.; Martinez, D. A.; McNaney, J. M.; Millot, M. A.; Pak, A. E.; Park, H. S.; Ping, Y.; Pollock, B. B.; Smith, R. F.; Wehrenberg, C. E.; Widmann, K.; Collins, G. W.; Landen, O. L.; Wan, A.; Hsing, W.

    2015-12-04

    In FY15, LLNL’s High-Energy-Density Physics (HED) and Indirect Drive Inertial Confinement Fusion (ICF-ID) programs conducted several campaigns on the OMEGA laser system and on the EP laser system, as well as campaigns that used the OMEGA and EP beams jointly. Overall these LLNL programs led 468 target shots in FY15, with 315 shots using just the OMEGA laser system, 145 shots using just the EP laser system, and 8 Joint shots using Omega and EP together. Approximately 25% of the total number of shots (56 OMEGA shots and 67 EP shots, including the 8 Joint shots) supported the Indirect Drive Inertial Confinement Fusion Campaign (ICF-ID). The remaining 75% (267 OMEGA shots and 86 EP shots) were dedicated to experiments for High-Energy-Density Physics (HED). Highlights of the various HED and ICF campaigns are summarized in the following reports.

  6. OMEGA-3 FATTY ACIDS IN CRITICAL ILLNESS

    PubMed Central

    Martin, Julie M.; Stapleton, Renee D.

    2015-01-01

    Supplementation of enteral nutritional formulas and parenteral nutrition lipid emulsions with omega-3 fatty acids is a recent area of research in patients with critical illness. It is hypothesized that omega-3 fatty acids may help reduce inflammation in critically ill patients, particularly those with sepsis and acute lung injury. The objective of this article is to review the data on supplementing omega-3 fatty acids during critical illness; enteral and parenteral supplementation are reviewed separately. The results of the research available to date are contradictory for both enteral and parenteral omega-3 fatty acid administration. Supplementation with omega-3 fatty acids may influence the acute inflammatory response in critically ill patients, but more research is needed before definitive recommendations about the routine use of omega-3 fatty acids in caring for critically ill patients can be made. PMID:20796218

  7. Studies of heat precipitable immunoglobulins

    PubMed Central

    Patterson, R.; Roberts, Mary; Pruzansky, J. J.

    1970-01-01

    The nature of the heat precipitation of 3 mononoclonal heat labile immunoglobulins was studied. These included 2 γG pyroglobulins and one γM pyroglobulin. Thermoprecipitable activity of both γG pyroglobulins could be localized to their heavy chains and to the Fab fragments of one of them. Heat precipitability of the γM paraprotein required the presence of the intact γM molecule since 7S subunits did not precipitate. The thermal precipitates appeared to result from intramolecular or intermolecular reactions with the formation of strong covalent bonds rather than weak non-covalent bonds. The importance of disulphide bonding was excluded in the precipitation of both γG but not in the γM pyroglobulins. Heat precipitation of the monoclonal γM resulted in coprecipitation of other proteins, particularly γG globulin, which suggested a specific type of reaction with this immunoglobulin. The interaction of the γM pyroglobulin, normal γG and heat produced an irreversible precipitate. ImagesFig. 1 PMID:4099668

  8. Integration of Omega and satellite navigation systems

    NASA Astrophysics Data System (ADS)

    Schlachta, Henry B.

    An extensive series of laboratory tests and flight trials has established that the hybrid Omega/VLF/GPS system effectively applies GPS to the enhancement of Omega with a cost-effective operator installation. The accuracy enhancement thus achieved also increases the reliability of navigation and furnishes aviation fuel savings superior to those of Omega, as a result of reduced flight-path wavering. The prospective GPS/GLONASS navigation system currently undergoing definition will be the first certifiable as a sole means on navigation; the Omega/VLF/GPS hybrid can serve as a transitional system.

  9. Using Caenorhabditis elegans to Uncover Conserved Functions of Omega-3 and Omega-6 Fatty Acids

    PubMed Central

    Watts, Jennifer L.

    2016-01-01

    The nematode Caenorhabditis elegans is a powerful model organism to study functions of polyunsaturated fatty acids. The ability to alter fatty acid composition with genetic manipulation and dietary supplementation permits the dissection of the roles of omega-3 and omega-6 fatty acids in many biological process including reproduction, aging and neurobiology. Studies in C. elegans to date have mostly identified overlapping functions of 20-carbon omega-6 and omega-3 fatty acids in reproduction and in neurons, however, specific roles for either omega-3 or omega-6 fatty acids are beginning to emerge. Recent findings with importance to human health include the identification of a conserved Cox-independent prostaglandin synthesis pathway, critical functions for cytochrome P450 derivatives of polyunsaturated fatty acids, the requirements for omega-6 and omega-3 fatty acids in sensory neurons, and the importance of fatty acid desaturation for long lifespan. Furthermore, the ability of C. elegans to interconvert omega-6 to omega-3 fatty acids using the FAT-1 omega-3 desaturase has been exploited in mammalian studies and biotechnology approaches to generate mammals capable of exogenous generation of omega-3 fatty acids. PMID:26848697

  10. Using Caenorhabditis elegans to Uncover Conserved Functions of Omega-3 and Omega-6 Fatty Acids.

    PubMed

    Watts, Jennifer L

    2016-02-02

    The nematode Caenorhabditis elegans is a powerful model organism to study functions of polyunsaturated fatty acids. The ability to alter fatty acid composition with genetic manipulation and dietary supplementation permits the dissection of the roles of omega-3 and omega-6 fatty acids in many biological process including reproduction, aging and neurobiology. Studies in C. elegans to date have mostly identified overlapping functions of 20-carbon omega-6 and omega-3 fatty acids in reproduction and in neurons, however, specific roles for either omega-3 or omega-6 fatty acids are beginning to emerge. Recent findings with importance to human health include the identification of a conserved Cox-independent prostaglandin synthesis pathway, critical functions for cytochrome P450 derivatives of polyunsaturated fatty acids, the requirements for omega-6 and omega-3 fatty acids in sensory neurons, and the importance of fatty acid desaturation for long lifespan. Furthermore, the ability of C. elegans to interconvert omega-6 to omega-3 fatty acids using the FAT-1 omega-3 desaturase has been exploited in mammalian studies and biotechnology approaches to generate mammals capable of exogenous generation of omega-3 fatty acids.

  11. High serum-free light chain levels and their rapid reduction in response to therapy define an aggressive multiple myeloma subtype with poor prognosis.

    PubMed

    van Rhee, Frits; Bolejack, Vanessa; Hollmig, Klaus; Pineda-Roman, Mauricio; Anaissie, Elias; Epstein, Joshua; Shaughnessy, John D; Zangari, Maurizio; Tricot, Guido; Mohiuddin, Abid; Alsayed, Yazan; Woods, Gail; Crowley, John; Barlogie, Bart

    2007-08-01

    Serum-free light chain (SFLC) levels are useful for diagnosing nonsecretory myeloma and monitoring response in light-chain-only disease, especially in the presence of renal failure. As part of a tandem autotransplantation trial for newly diagnosed multiple myeloma, SFLC levels were measured at baseline, within 7 days of starting the first cycle, and before both the second induction cycle and the first transplantation. SFLC baseline levels higher than 75 mg/dL (top tertile) identified 33% of 301 patients with higher near-complete response rate (n-CR) to induction therapy (37% vs 20%, P = .002) yet inferior 24-month overall survival (OS: 76% vs 91%, P < .001) and event-free survival (EFS: 73% vs 90%, P < .001), retaining independent prognostic significance for both EFS (HR = 2.40, P = .008) and OS (HR = 2.43, P = .016). Baseline SFLC higher than 75 mg/dL was associated with light-chain-only secretion (P < .001), creatinine level 176.8 microM (2 mg/dL) or higher (P < .001), beta-2-microglobulin 297.5 nM/L (3.5 mg/L) or higher (P < .001), lactate dehydrogenase 190 U/L or higher (P < .001), and bone marrow plasmacytosis higher than 30% (P = .003). Additional independent adverse implications were conferred by top-tertile SFLC reductions before cycle 2 (OS: HR = 2.97, P = .003; EFS: HR = 2.56, P = .003) and before transplantation (OS: HR = 3.31, P = .001; EFS: HR = 2.65, P = .003). Unlike baseline and follow-up analyses of serum and urine M-proteins, high SFLC levels at baseline-reflecting more aggressive disease-and steeper reductions after therapy identified patients with inferior survival.

  12. Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: insights from x-ray crystallography

    DOE PAGES

    Kumaran, D.; Adler, M.; Levit, M.; Krebs, M.; Sweeney, R.; Swaminathan, S.

    2015-10-17

    The seven antigenically distinct serotypes (A to G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins: the heavy chain is responsible for neurospecific binding, internalization and translocation, and the light chain is responsible for substrate cleavage. Because of their extreme toxicity and prior history of weaponization, the BoNTs are considered to be potential bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than critical care;more » therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was synthesized and found to inhibit BoNT/A light chain (Balc) with high affinity. When tested in a cell-free Förster resonance excitation transfer (FRET) assay, CPI-1 was found to have a Ki of 13.9 nM using full-length Balc448 and 42.1 nM using a truncated crystallizable form of light chain (Balc424). Co-crystallization of CPI-1 with Balc424 revealed that in the Balc-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn2+ binding region involved in catalysis. This is in contrast to linear peptide inhibitors described to date which block only the latter« less

  13. Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: insights from x-ray crystallography

    SciTech Connect

    Kumaran, D.; Adler, M.; Levit, M.; Krebs, M.; Sweeney, R.; Swaminathan, S.

    2015-10-17

    The seven antigenically distinct serotypes (A to G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins: the heavy chain is responsible for neurospecific binding, internalization and translocation, and the light chain is responsible for substrate cleavage. Because of their extreme toxicity and prior history of weaponization, the BoNTs are considered to be potential bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than critical care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was synthesized and found to inhibit BoNT/A light chain (Balc) with high affinity. When tested in a cell-free Förster resonance excitation transfer (FRET) assay, CPI-1 was found to have a Ki of 13.9 nM using full-length Balc448 and 42.1 nM using a truncated crystallizable form of light chain (Balc424). Co-crystallization of CPI-1 with Balc424 revealed that in the Balc-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn2+ binding region involved in catalysis. This is in contrast to linear peptide inhibitors described to date which block only the latter

  14. Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: Insights from X-ray crystallography.

    PubMed

    Kumaran, Desigan; Adler, Michael; Levit, Matthew; Krebs, Michael; Sweeney, Richard; Swaminathan, Subramanyam

    2015-11-15

    The seven antigenically distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins consisting of a ∼ 100 kDa heavy chain and a ∼ 50 kDa light chain; the former is responsible for neurospecific binding, internalization and translocation, and the latter for cleavage of neuronal SNARE proteins. Because of their extreme toxicity and history of weaponization, the BoNTs are regarded as potential biowarfare/bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than intensive care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was evaluated in a FRET assay for its ability to inhibit BoNT/A light chain (Balc). CPI was found to be highly potent, exhibiting a Ki of 12.3 nM with full-length Balc448 and 39.2 nM using a truncated crystallizable form of the light chain (Balc424). Cocrystallization studies revealed that in the Balc424-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn(2+) binding region involved in catalysis. This differs from linear peptide inhibitors described to date which block only the latter. PMID:26522088

  15. Prognostic utility of intact immunoglobulin Ig′κ/Ig′λ ratios in multiple myeloma patients

    PubMed Central

    Bradwell, A; Harding, S; Fourrier, N; Mathiot, C; Attal, M; Moreau, P; Harousseau, J-L; Avet-Loiseau, H

    2013-01-01

    To determine whether isotype matched immunoglobulin (Ig; Ig′κ/Ig′λ) ratios had prognostic significance in patients with intact Ig multiple myeloma (MM). Novel immunoassays measuring serum concentrations of the Ig heavy chain/light chain (HLC) subsets IgGκ, IgGλ, IgAκ and IgAλ were compared with monoclonal protein (‘M-spike') quantification by serum protein electrophoresis, β2-microglobulin (β2-M), albumin, serum free light chain (FLC) and cytogenetic markers in relation to outcome in 339 MM patients. Abnormal IgGκ/IgGλ and IgAκ/IgAλ ratios present in the respective tumor isotypes at clinical presentation were predictive of shorter progression-free survival (PFS) (hazard ratio (HR) 1.9; P=0.0002), predominantly due to the suppression of the uninvolved (polyclonal) Ig of the same isotype as the tumor (HR 1.8; P=0.002). No significant associations were observed between PFS and M-spike concentrations, suppression of non-tumor Igs of different isotypes or FLC κ/λ ratios. β2-M and HLC ratios were independently prognostic (P=0.045 and P=0.001). A staging system using β2-M and extreme HLC ratios (<0.01 or >200) had greater prognostic value than the widely used ISS staging system (HR 1.7; P=0.00002 vs HR 1.3; P=0.017). These results suggest that HLC ratios may have a role in clinical management of MM. PMID:22699454

  16. The Challenges of omega Centauri

    NASA Astrophysics Data System (ADS)

    Norris, John E.

    2012-05-01

    It is now five decades since the demonstration that the color magnitude diagram (CMD) of omega Cen is unlike that of other globular clusters. This was followed, slowly but surely, by the appreciation that omega contains a large number (some five and counting) of discrete populations. The challenge being addressed is the determination of the spatial, chemical abundance, kinematic, and age distributions of these populations, and their inter-relationships - which will constrain our understanding of the origin and evolution of the system. The present contribution addresses the problems currently posed by our interpretation of the existing data set. With exquisite photometry, discrete sequences are now clearly seen in the CMD well down onto the main sequence. This is accompanied by high-resolution spectroscopy (albeit not to such faint levels), permitting insight into the abundance and kinematic distributions of large samples. Ages, however, are very difficult to determine, and those available defy simple interpretation in terms of population parameters. Major problems are now evident. Consider the multiple main sequence: if this results from postulated large helium abundance differences (Y in the range 0.25 - 0.40), what is the origin of the extremely high values. Is it really helium that is varying?! Second, one needs a reconciliation of the CMD sequences in terms of the abundances (He, CNO and Fe) and ages of the relevant populations. How do three populations on the main sequence telescope to five (or more) on the post main sequence giant branches? Where do the populations evolve onto the horizontal branch and the white dwarf sequence? What drives variations in population parameters with distance from cluster center? Finally, if omega Cen is the remnant of a nucleated dwarf galaxy captured long ago by the Milky Way, where is the debris of that event among the Galaxy's field halo population?

  17. Phosphorylation by protein kinase C of the 20,000-dalton light chain of myosin in intact and chemically skinned vascular smooth muscle.

    PubMed

    Sutton, T A; Haeberle, J R

    1990-02-15

    In the present study we tested the hypothesis that phosphorylation of the 20,000-dalton light chain subunit of smooth muscle myosin (LC20) by the calcium-activated and phospholipid-dependent protein kinase C regulates contraction of chemically-permeabilized (glycerinated) porcine carotid artery smooth muscle. Purified protein kinase C and oleic acid were used to phosphorylate LC20 in glycerinated muscles in the presence of a CaEGTA/EGTA buffer system (pCa 8) to prevent activation of myosin light chain kinase. Phosphorylation of the light chain to 1.3 mol of PO4/mol of LC20 did not stimulate contraction. Tryptic digests of glycerinated carotid artery LC20 contained two major phosphopeptides which contained phosphoserine but not phosphothreonine. Incubation of glycerinated muscles with calcium (20 microM) and calmodulin (10 microM) resulted in contraction and LC20 phosphorylation to 1.1 mol of PO4/mol of LC20; tryptic digests of LC20 from these muscles contained a single phosphopeptide which could be distinguished by phosphopeptide mapping from the two phosphopeptides derived from muscles phosphorylated with protein kinase C. Further phosphorylation of Ca2+/calmodulin-activated muscles to 2.0 mol of PO4/mol of LC20, by incubation with protein kinase C, had no effect on either the level of isometric force or the lightly-loaded shortening velocity (after-load = 0.1 peak active force); removal of Ca2+ and calmodulin, but not protein kinase C and oleic acid, resulted in normal relaxation in spite of maintained phosphorylation to 1.2 mol of PO4/mol of LC20. Comparison of LC20 phosphopeptide maps from glycerinated muscles incubated with protein kinase C plus Ca2+/calmodulin (2.0 mol of PO4/mol of LC20) to maps from intact muscles stimulated with 10(-6) M phorbol 12,13-dibutyrate (0.05 mol of PO4/mol of LC20) showed that the same three phosphopeptides were present in both the intact and glycerinated muscles. These findings show that phosphorylation of LC20 by protein kinase

  18. Perspectives on Immunoglobulins in Colostrum and Milk

    PubMed Central

    Hurley, Walter L.; Theil, Peter K.

    2011-01-01

    Immunoglobulins form an important component of the immunological activity found in milk and colostrum. They are central to the immunological link that occurs when the mother transfers passive immunity to the offspring. The mechanism of transfer varies among mammalian species. Cattle provide a readily available immune rich colostrum and milk in large quantities, making those secretions important potential sources of immune products that may benefit humans. Immune milk is a term used to describe a range of products of the bovine mammary gland that have been tested against several human diseases. The use of colostrum or milk as a source of immunoglobulins, whether intended for the neonate of the species producing the secretion or for a different species, can be viewed in the context of the types of immunoglobulins in the secretion, the mechanisms by which the immunoglobulins are secreted, and the mechanisms by which the neonate or adult consuming the milk then gains immunological benefit. The stability of immunoglobulins as they undergo processing in the milk, or undergo digestion in the intestine, is an additional consideration for evaluating the value of milk immunoglobulins. This review summarizes the fundamental knowledge of immunoglobulins found in colostrum, milk, and immune milk. PMID:22254105

  19. Antibody light-chain-restricted recognition of the site of immune pressure in the RV144 HIV-1 vaccine trial is phylogenetically conserved.

    PubMed

    Wiehe, Kevin; Easterhoff, David; Luo, Kan; Nicely, Nathan I; Bradley, Todd; Jaeger, Frederick H; Dennison, S Moses; Zhang, Ruijun; Lloyd, Krissey E; Stolarchuk, Christina; Parks, Robert; Sutherland, Laura L; Scearce, Richard M; Morris, Lynn; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Sinangil, Faruk; Phogat, Sanjay; Michael, Nelson L; Kim, Jerome H; Kelsoe, Garnett; Montefiori, David C; Tomaras, Georgia D; Bonsignori, Mattia; Santra, Sampa; Kepler, Thomas B; Alam, S Munir; Moody, M Anthony; Liao, Hua-Xin; Haynes, Barton F

    2014-12-18

    In HIV-1, the ability to mount antibody responses to conserved, neutralizing epitopes is critical for protection. Here we have studied the light chain usage of human and rhesus macaque antibodies targeted to a dominant region of the HIV-1 envelope second variable (V2) region involving lysine (K) 169, the site of immune pressure in the RV144 vaccine efficacy trial. We found that humans and rhesus macaques used orthologous lambda variable gene segments encoding a glutamic acid-aspartic acid (ED) motif for K169 recognition. Structure determination of an unmutated ancestor antibody demonstrated that the V2 binding site was preconfigured for ED motif-mediated recognition prior to maturation. Thus, light chain usage for recognition of the site of immune pressure in the RV144 trial is highly conserved across species. These data indicate that the HIV-1 K169-recognizing ED motif has persisted over the diversification between rhesus macaques and humans, suggesting an evolutionary advantage of this antibody recognition mode.

  20. Carbachol ameliorates lipopolysaccharide-induced intestinal epithelial tight junction damage by down-regulating NF-{kappa}{beta} and myosin light-chain kinase pathways

    SciTech Connect

    Zhang, Ying; Li, Jianguo

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Carbachol reduced the lipopolysaccharide-induced intestinal barrier breakdown. Black-Right-Pointing-Pointer Carbachol ameliorated the lipopolysaccharide-induced ileal tight junction damage. Black-Right-Pointing-Pointer Carbachol prevented the LPS-induced NF-{kappa}{beta} and myosin light-chain kinase activation. Black-Right-Pointing-Pointer Carbachol exerted its beneficial effects in an {alpha}7 nicotinic receptor-dependent manner. -- Abstract: Carbachol is a cholinergic agonist that protects the intestines after trauma or burn injury. The present study determines the beneficial effects of carbachol and the mechanisms by which it ameliorates the lipopolysaccharide (LPS)-induced intestinal barrier breakdown. Rats were injected intraperitoneally with 10 mg/kg LPS. Results showed that the gut barrier permeability was reduced, the ultrastructural disruption of tight junctions (TJs) was prevented, the redistribution of zonula occludens-1 and claudin-2 proteins was partially reversed, and the nuclear factor-kappa beta (NF-{kappa}{beta}) and myosin light-chain kinase (MLCK) activation in the intestinal epithelium were suppressed after carbachol administration in LPS-exposed rats. Pretreatment with the {alpha}7 nicotinic acetylcholine receptor ({alpha}7nAchR) antagonist {alpha}-bungarotoxin blocked the protective action of carbachol. These results suggested that carbachol treatment can protect LPS-induced intestinal barrier dysfunction. Carbachol exerts its beneficial effect on the amelioration of the TJ damage by inhibiting the NF-{kappa}{beta} and MLCK pathways in an {alpha}7nAchR-dependent manner.

  1. Molecular cloning and nucleotide sequences of the complementary DNAs to chicken skeletal muscle myosin two alkali light chain mRNAs.

    PubMed Central

    Nabeshima, Y; Fujii-Kuriyama, Y; Muramatsu, M; Ogata, K

    1982-01-01

    We report here the molecular cloning and sequence analysis of DNAs complementary to mRNAs for myosin alkali light chain of chicken embryo and adult leg skeletal muscle. pSMA2-1 contained an 818 base-pair insert that includes the entire coding region and 5' and 3' untranslated regions of A2 mRNA. pSMA1-1 contained a 848 base-pair insert that included the 3' untranslated region and almost all of the coding region except for the N-terminal 13 amino acid residues of the A1 light chain. The 741 nucleotide sequences of A1 and A2 mRNAs corresponding to C-terminal 141 amino acid residues and 3' untranslated regions were identical. The 5' terminal nucleotide sequences corresponding to N-terminal 35 amino acid residues of A1 chain were quite different from the sequences corresponding to N-terminal 8 amino acid residues and of the 5' untranslated region of A2 mRNA. These findings are discussed in relation to the structures of the genes for A1 and A2 mRNA. PMID:6128725

  2. A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity.

    PubMed

    Heaslip, Aoife T; Leung, Jacqueline M; Carey, Kimberly L; Catti, Federica; Warshaw, David M; Westwood, Nicholas J; Ballif, Bryan A; Ward, Gary E

    2010-01-15

    Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains.

  3. Clock synchronization experiments using OMEGA transmissions

    NASA Technical Reports Server (NTRS)

    Chi, A. R.; Wardrip, S. C.

    1973-01-01

    The OMEGA transmissions from North Dakota on 13.10 and 12.85 kHz were monitored at several sites using a recently developed OMEGA timing receiver specifically designed for this purpose. The experiments were conducted at Goddard Space Flight Center, Greenbelt, Maryland; U.S. Naval Observatory, Washington, D.C.; and at the NASA tracking station, Rosman, North Carolina. Results show that cycle identification of the two carrier frequencies was made at each test site, thus, coarse time (76 microseconds) from the OMEGA transmitted signals to within the ambiguity period of each OMEGA frequency was extracted. The fine time determination, which was extracted from the phase difference between the received OMEGA signals and locally generated signals, was about + or - 2 microseconds for daytime reception and about + or - 5 microseconds for nighttime reception.

  4. PHYLOGENY OF IMMUNOGLOBULIN STRUCTURE AND FUNCTION

    PubMed Central

    Clem, L. W.; Small, P. A.

    1967-01-01

    Lemon sharks immunized with bovine serum albumin produced two molecular forms of antibodies detectable by passive hemagglutination of antigen-coated, tanned sheep erythrocytes. Throughout the course of immunization 2-ME-sensitive antibody was associated with a 19S immunoglobulin fraction (4–5 mg/ml serum) while late in the course of immunization antibody was found also associated with a 7S immunoglobulin fraction (7–8 mg/ml serum). No evidence for any anamnestic response was found in these animals. Naturally occurring hemagglutinins for sheep erythrocytes were found to be 2-ME-sensitive and present in the 19S immunoglobulin fraction. These immunoglobulin fractions were readily purified by DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Both immunoglobulin molecules yielded equimolar amounts of H and L polypeptide chains when subjected to extensive reduction and alkylation followed by gel filtration in 5 M guanidine-HCl. Antigenically reactive H and L chains were obtained by partial reduction and alkylation followed by gel filtration in 1 M propionic acid. The 7S and 19S immunoglobulin H chains were indistinguishable by fingerprints of tryptic digests, disc electrophoretic patterns, antigenic properties, and mass (molecular weight ∼70,000), thus suggesting these two molecules to belong to the same immunoglobulin class. The shark 19S and 7S immunoglobulin L chains were indistinguishable from each other by similar criteria and were different from the H chains. These L chains exhibited the electrophoretic heterogeneity of their mammalian counterparts. The 7S (shark immunoglobulin) molecule was shown to have a molecular weight of ∼160,000 and to consist of 2H and 2L polypeptide chains (total mass ≅180,000). The 19S molecule was shown to have a molecular weight of 800,000–900,000; therefore, there were probably five 7S subunits per 19S molecule, comparable to mammalian γM. Other reasons for considering the 7S and the 19S lemon shark

  5. Optics Performance at 1(omega), 2 (omega), and 3 (omega): Final Report on LDRD Project 03-ERD-071

    SciTech Connect

    Honig, J; Adams, J; Carr, C; Demos, S; Feit, M; Mehta, N; Norton, M; Nostrand, M; Rubenchik, A; Spaeth, M

    2006-02-08

    The interaction of intense laser light with dielectric materials is a fundamental applied science problem that is becoming increasingly important with the rapid development of ever more powerful lasers. To better understand the behavior of optical components in large fusion-class laser systems, we are systematically studying the interaction of high-fluence, high-power laser light with high-quality optical components, with particular interest on polishing/finishing and stress-induced defects and surface contamination. We focus on obtaining comparable measurements at three different wavelengths, 1{omega} (1053 nm), 2{omega} (527 nm), and 3{omega} (351 nm).

  6. [Cardiovascular disease and omega-3 fatty acids].

    PubMed

    Ponte, E; Cafagna, D; Balbi, M

    1997-09-01

    Fish oil is rich in the long chain omega-3 (omega-3) polyinsaturated fatty acids (PUFA), Pioneering studies of Dyerberg and Bang primarily originate interests in this way. The low incidence of acute myocardial infarction they verified within the Greenland Eskimos suggested that a high dietary omega-3 PUFA intake due to marine food might protect against coronary heart disease. They showed that the Eskimos had a beneficial lipid pattern and that their balance between pro-aggregatory thromboxanes and anti-aggregatory prostacyclins was shifted towards an anti-thrombotic state. The two major omega-3 fatty acids are decosapentaenoic acid (EPA C 20:5, omega 3), with five double bonds, and docosahexaenoic acid (DHA C 22:6, omega 3), with six double bonds. These fatty acids' significant effects include reduction of plasma triglycerides and lipoprotein levels as well as of platelets thrombogenicity in the microcirculation, which is due to effects on the mediators production derived from arachidonic acid (prostaglandins and leucotrienes), meddling in inflammatory and immune cell function, retarded atherosclerosis development. Experimental studies of atherogenesis and arterial thrombogenesis support the hypothesis that dietary omega-3 PUFA intake may play a leading role in primary or secondary prevention of coronary heart disease.

  7. Functional and safety evaluation of transgenic pork rich in omega-3 fatty acids.

    PubMed

    Tang, Maoxue; Qian, Lili; Jiang, Shengwang; Zhang, Jian; Song, Pengkun; Chen, Yaoxing; Cui, Wentao; Li, Kui

    2014-08-01

    Genetically modified animals rich in omega-3 unsaturated fatty acid offer a new strategy to improve the human health, but at the same time present a challenge in terms of food safety assessment. In this study, we evaluated the function and safety of sFat-1 transgenic pork rich in omega-3 fatty acids in mice by feeding basic diet and diets that contain wild type pork and sFat-1 transgenic pork. Blood biochemistry, haematology, peripheral T cell distributions, bacterial counts, gross necropsy, histopathology and organ weights were performed in mice fed with different doses of wild type and transgenic pork. Results indicated that both low and high dose of wild type and transgenic pork had no significant effect on blood biochemistry, T cell distribution, immunoglobulins and bacterial counts in intestine and feces. However, it was noted that both low and high dose of transgenic pork improved the liver immune system in mice, which is probably due to the beneficial contribution of high level of the "good" fatty acids in transgenic pork. There is no significant effect of transgenic pork on all other organs in mice. In summary, our study clearly demonstrated that feeding transgenic pork rich in omega-3 fatty acids did not cause any harm to mice, and in fact, improved the liver immune system.

  8. Cytomegalovirus Immunoglobulin After Thoracic Transplantation

    PubMed Central

    Grossi, Paolo; Mohacsi, Paul; Szabolcs, Zoltán; Potena, Luciano

    2016-01-01

    Abstract Cytomegalovirus (CMV) is a highly complex pathogen which, despite modern prophylactic regimens, continues to affect a high proportion of thoracic organ transplant recipients. The symptomatic manifestations of CMV infection are compounded by adverse indirect effects induced by the multiple immunomodulatory actions of CMV. These include a higher risk of acute rejection, cardiac allograft vasculopathy after heart transplantation, and potentially bronchiolitis obliterans syndrome in lung transplant recipients, with a greater propensity for opportunistic secondary infections. Prophylaxis for CMV using antiviral agents (typically oral valganciclovir or intravenous ganciclovir) is now almost universal, at least in high-risk transplants (D+/R−). Even with extended prophylactic regimens, however, challenges remain. The CMV events can still occur despite antiviral prophylaxis, including late-onset infection or recurrent disease, and patients with ganciclovir-resistant CMV infection or who are intolerant to antiviral therapy require alternative strategies. The CMV immunoglobulin (CMVIG) and antiviral agents have complementary modes of action. High-titer CMVIG preparations provide passive CMV-specific immunity but also exert complex immunomodulatory properties which augment the antiviral effect of antiviral agents and offer the potential to suppress the indirect effects of CMV infection. This supplement discusses the available data concerning the immunological and clinical effects of CMVIG after heart or lung transplantation. PMID:26900989

  9. LC8 dynein light chain (DYNLL1) binds to the C-terminal domain of ATM-interacting protein (ATMIN/ASCIZ) and regulates its subcellular localization

    SciTech Connect

    Rapali, Peter; Garcia-Mayoral, Maria Flor; Martinez-Moreno, Monica; Tarnok, Krisztian; Schlett, Katalin; Albar, Juan Pablo; Bruix, Marta; Nyitray, Laszlo; Rodriguez-Crespo, Ignacio

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer We have screened a human library with dynein light chain DYNLL1 (DLC8) as bait. Black-Right-Pointing-Pointer Dynein light chain DYNLL1 binds to ATM-kinase interacting protein (ATMIN). Black-Right-Pointing-Pointer ATMIN has 17 SQ/TQ motifs, a motif frequently found in DYNLL1-binding partners. Black-Right-Pointing-Pointer The two proteins interact in vitro, with ATMIN displaying at least five binding sites. Black-Right-Pointing-Pointer The interaction of ATMIN and DYNNL1 in transfected cells can also be observed. -- Abstract: LC8 dynein light chain (now termed DYNLL1 and DYNLL2 in mammals), a dimeric 89 amino acid protein, is a component of the dynein multi-protein complex. However a substantial amount of DYNLL1 is not associated to microtubules and it can thus interact with dozens of cellular and viral proteins that display well-defined, short linear motifs. Using DYNLL1 as bait in a yeast two-hybrid screen of a human heart library we identified ATMIN, an ATM kinase-interacting protein, as a DYNLL1-binding partner. Interestingly, ATMIN displays at least 18 SQ/TQ motifs in its sequence and DYNLL1 is known to bind to proteins with KXTQT motifs. Using pepscan and yeast two-hybrid techniques we show that DYNLL1 binds to multiple SQ/TQ motifs present in the carboxy-terminal domain of ATMIN. Recombinant expression and purification of the DYNLL1-binding region of ATMIN allowed us to obtain a polypeptide with an apparent molecular mass in gel filtration close to 400 kDa that could bind to DYNLL1 in vitro. The NMR data-driven modelled complexes of DYNLL1 with two selected ATMIN peptides revealed a similar mode of binding to that observed between DYNLL1 and other peptide targets. Remarkably, co-expression of mCherry-DYNLL1 and GFP-ATMIN mutually affected intracellular protein localization. In GFP-ATMIN expressing-cells DNA damage induced efficiently nuclear foci formation, which was partly impeded by the presence of mCherry-DYNLL1

  10. [Immunoglobulin genes encoding antibodies directed to oncodevelopmental carbohydrate antigens].

    PubMed

    Zenita, K; Yago, K; Fujimoto, E; Kannagi, R

    1990-07-01

    We investigated the immunoglobulin genes which encode the variable region of the monoclonal antibodies directed to the onco-developmental carbohydrate antigens such SSEA-1, fucosyl SSEA-1, SSEA-3 and SSEA-4. The VH region of these antibodies was preferentially encoded by the gene members of the X24, VH7183 and Q52 families, the families which are known to be located at the 3'-end region of the murine germ line VH gene. This result is interesting particularly when considering that the members of the 3'-end VH families are known to be preferentially expressed in embryonic B lymphocytes by an intrinsic genetic program. The comparative study of the nucleic acid sequences of mRNAs encoding these antibodies and the sequences of the corresponding germ line VH genes disclosed that the sequences encoding the antibodies contain no mutation from the germ line VH genes, or contain only a few somatic mutations, which are thought to be insignificant for the reactivity of the antibodies to the nominal antigens. These results imply that some of the embryonic B lymphocytes that express the unmutated germ line VH genes of the 3'-end families can be reactive with embryonic carbohydrate antigens, albeit rearranged with appropriate D-JH gene segments, and coupled with proper light chains. The VH region of the syngenic monoclonal anti-idiotypic antibodies directed to these anti-carbohydrate antibodies were also encoded preferentially by the members of the 3'-end VH families. We propose here that a part of the virgin embryonic B lymphocytes, which express the antibody encoded by the gene members of the 3'-end VH families at the cell surface, will be stimulated by the embryonic carbohydrate antigens which are abundantly present in the internal milieu of the embryo. The clonally expanded B lymphocytes, in turn, will facilitate the proliferation of other populations of embryonic B lymphocytes expressing the corresponding anti-idiotypic antibodies, which are also encoded by the gene members

  11. Mars Express OMEGA Observations over Terra Meridiani

    NASA Technical Reports Server (NTRS)

    Arvidson, R. E.; Poulet, F.; Bibring, J.-P.; Wolff, M.; Gendrin, A.; Morris, R. V.; Freeman, J. J.; Bellucci, G.

    2005-01-01

    The OMEGA hyperspectral imager (0.35 to 5.08 micrometers) covered the hematite-bearing plains and underlying etched terrains of Terra Meridiani during several orbits with spatial resolutions ranging from several hundred meters to approximately 2 km. We extracted and analyzed surface reflectance spectra from OMEGA data for the approximately 864,000 square kilometers surrounding the Opportunity site. In this paper we focus on analysis of OMEGA orbit 485 data for the plains and etched terrains located to the northeast of the Opportunity landing site.

  12. Polar-Direct-Drive Experiments on OMEGA

    SciTech Connect

    Marshall, F.J.; Craxton, R.S.; Bonino, M.J.; Epstein, R.; Glebov, V.Yu.; Jacobs-Perkins, D.; Knauer, J.P.; Marozas, J.A.; McKenty, P.W.; Noyes, S.G.; Radha, P.B.; Seka, W.; Skupsky, S.; Smalyuk

    2006-06-28

    Polar direct drive (PDD), a promising ignition path for the NIF while the beams are in the indirect-drive configuration, is currently being investigated on the OMEGA laser system by using 40 beams in six rings repointed to more uniformly illuminate the target. The OMEGA experiments are being performed with standard, “warm” targets with and without the use of an equatorial “Saturn-like” toroidally shaped CH ring. Target implosion symmetry is diagnosed with framed x-ray backlighting using additional OMEGA beams and by time-integrated x-ray imaging of the stagnating core.

  13. Omega-3 and omega-6 fatty acids and type 2 diabetes.

    PubMed

    Jeppesen, Charlotte; Schiller, Katja; Schulze, Matthias B

    2013-04-01

    Polyunsaturated fatty acids are of particular interest in the nutritional therapy for diabetes, given their potential role in several pathophysiological processes related to cardiovascular disease. Both omega-3 and omega-6 fatty acids are beneficial for improving lipid profiles in healthy individuals and among type 2 diabetic patients: Supplementation with omega-3 fatty acids lowers triglycerides and VLDL-cholesterol. However, they might also increase LDL-cholesterol. Omega-3 fatty acids are, from the latest evidence, not related to mortality and cardiovascular disease. Similarly, glucose control and hypertension, as well as risk of microvascular complications, seem unaffected by omega-3 supplementation. Most studies involved mainly patients with type 2 diabetes, and future research needs to focus on the type 1 diabetic patient. Also, the role of omega-6 fatty acids remains largely unknown.

  14. Identification of the t Complex–encoded Cytoplasmic Dynein Light Chain Tctex1 in Inner Arm I1 Supports the Involvement of Flagellar Dyneins in Meiotic Drive

    PubMed Central

    Harrison, Alistair; Olds-Clarke, Patricia; King, Stephen M.

    1998-01-01

    The cytoplasmic dynein light chain Tctex1 is a candidate for one of the distorter products involved in the non-Mendelian transmission of mouse t haplotypes. It has been unclear, however, how the t-specific mutations in this protein, which is found associated with cytoplasmic dynein in many tissues, could result in a male germ cell–specific phenotype. Here, we demonstrate that Tctex1 is not only a cytoplasmic dynein component, but is also present both in mouse sperm and Chlamydomonas flagella. Genetic and biochemical dissection of the Chlamydomonas flagellum reveal that Tctex1 is a previously undescribed component of inner dynein arm I1. Combined with the recent identification of another putative t complex distorter, Tctex2, within the outer dynein arm, these results support the hypothesis that transmission ratio distortion (meiotic drive) of mouse t haplotypes involves dysfunction of both flagellar inner and outer dynein arms but does not require the cytoplasmic isozyme. PMID:9490726

  15. Tumor necrosis factor and immune interferon synergistically increase transcription of HLA class I heavy- and light-chain genes in vascular endothelium

    SciTech Connect

    Johnson, D.R.; Pober, J.S. )

    1990-07-01

    Tumor necrosis factor and immune interferon synergistically increase cell-surface expression of class I major histocompatibility complex molecules in cultured human endothelial cells. The authors report that tumor necrosis factor and interferon {gamma} each independently increase mRNA levels and together cause a greater-than-additive (i.e., synergistic) increase in steady-state mRNA levels and transcriptional rates of the class I heavy- and light-chain genes. HLA heavy-chain mRNA is equally stable in cytokine-treated and -untreated endothelial cells. Interferon {gamma} does not increase tumor necrosis factor receptor number or affinity on human endothelial cells. They conclude that the synergistic increase in class I major histocompatibility complex cell-surface expression results principally from the synergistic increase in transcriptional rates. They propose that this increase is caused by the cooperative binding of independently activated transcription factors to the promoter/enhancer sequences of class I genes.

  16. Ratio of involved/uninvolved immunoglobulin quantification by Hevylite™ assay: clinical and prognostic impact in multiple myeloma

    PubMed Central

    2012-01-01

    Background HevyLite™ is a new, recently developed method that facilitates separate quantification of the kappa- and lambda-bounded amounts of a given immunoglobulin (Ig). Using this method, we measured intact immunoglobulin (heavy/light chain; HLC) IgG-kappa, IgG-lambda, IgA-kappa, IgA-lambda individually, as well as their deriving ratios (HLCR) in a series of IgG or IgA multiple myeloma (MM) patients, to investigate and assess the contribution of these tests to disease evaluation. Patients and methods HevyLite™ assays were used in sera from 130 healthy individuals (HI) and 103 MM patients, at time of diagnosis. In patients, the level of paraprotein was IgG in 78 (52 IgG-kappa, 26 IgG-lambda) and IgΑ in 25 (13 IgΑ-kappa, 12 IgΑ-lambda). Durie-Salmon and International Staging System stages were evenly distributed. Symptomatic patients (n = 77) received treatment while asymptomatic ones (n = 26) were followed. Patients' median follow-up was at 32.6 months. HLCR was calculated with the involved Ig (either G or A) as numerator. Results In HI, median IgG-kappa was 6.85, IgG-lambda 3.81, IgA-kappa 1.19 and IgA-lambda 0.98 g/L. The corresponding median involving HLC values in MM patients were 25.8, 23.45, 28.9 and 36.4 g/L. HLC-IgG related to anemia, high serum free light chain ratio and extensive bone marrow infiltration, while high HLCR correlated with the same plus increased β2-microglobulin. In addition, increased HLCR and the presence of immunoparesis correlated with time to treatment. Patients with high HLCR had a significantly shorter survival (p = 0.022); HLCR retained its prognostic value in multivariate analysis. Conclusions HLC and HLCR quantify the precise amount of the involved immunoglobulin more accurately than other methods; moreover, they carry prognostic information regarding survival in MM patients. PMID:23211046

  17. L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins.

    PubMed

    Maddala, Rupalatha; Nagendran, Tharkika; de Ridder, Gustaaf G; Schey, Kevin L; Rao, Ponugoti Vasantha

    2013-01-01

    Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial

  18. Aurora B but Not Rho/MLCK Signaling Is Required for Localization of Diphosphorylated Myosin II Regulatory Light Chain to the Midzone in Cytokinesis

    PubMed Central

    Kondo, Tomo; Isoda, Rieko; Ookusa, Takayuki; Kamijo, Keiju; Hamao, Kozue; Hosoya, Hiroshi

    2013-01-01

    Non-muscle myosin II is stimulated by monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC) further enhances the ATPase activity of myosin II. Phosphorylated MRLCs localize to the contractile ring and regulate cytokinesis as subunits of activated myosin II. Recently, we reported that 2P-MRLC, but not 1P-MRLC, localizes to the midzone independently of myosin II heavy chain during cytokinesis in cultured mammalian cells. However, the mechanism underlying the distinct localization of 1P- and 2P-MRLC during cytokinesis is unknown. Here, we showed that depletion of the Rho signaling proteins MKLP1, MgcRacGAP, or ECT2 inhibited the localization of 1P-MRLC to the contractile ring but not the localization of 2P-MRLC to the midzone. In contrast, depleting or inhibiting a midzone-localizing kinase, Aurora B, perturbed the localization of 2P-MRLC to the midzone but not the localization of 1P-MRLC to the contractile ring. We did not observe any change in the localization of phosphorylated MRLC in myosin light-chain kinase (MLCK)-inhibited cells. Furrow regression was observed in Aurora B- and 2P-MRLC-inhibited cells but not in 1P-MRLC-perturbed dividing cells. Furthermore, Aurora B bound to 2P-MRLC in vitro and in vivo. These results suggest that Aurora B, but not Rho/MLCK signaling, is essential for the localization of 2P-MRLC to the midzone in dividing HeLa cells. PMID:23951055

  19. Macromolecular substrate-binding exosites on both the heavy and light chains of factor XIa mediate the formation of the Michaelis complex required for factor IX-activation.

    PubMed

    Sinha, Dipali; Marcinkiewicz, Mariola; Navaneetham, Duraiswamy; Walsh, Peter N

    2007-08-28

    Binding of factor IX (FIX) to an exosite on the heavy chain of factor XIa (FXIa) is essential for the optimal activation of FIX (Sinha, D., Seaman, F. S., and Walsh, P. N. (1987) Biochemistry 26, 3768-3775). To gain further insight into the mechanisms of activation of FIX by FXIa, we have investigated the kinetic properties of FXIa-light chain (FXIa-LC) with its active site occupied by either a reversible inhibitor of serine proteases (p-aminobenzamidine, PAB) or a small peptidyl substrate (S-2366) and have examined FIX cleavage products resulting from activation by FXIa or FXIa-LC. PAB inhibited the hydrolysis of S-2366 by FXIa-LC in a classically competitive fashion. In contrast, PAB was found to be a noncompetitive inhibitor of the activation of the macromolecular substrate FIX. Occupancy of the active site of the FXIa-LC by S-2366 also resulted in noncompetitive inhibition of FIX activation. These results demonstrate the presence of an exosite for FIX binding on the FXIa-LC remote from its active site. Furthermore, examination of the cleavage products of FIX indicated that in the absence of either Ca2+ or the heavy chain of FXIa there was substantial accumulation of the inactive intermediate FIXalpha, indicating a slower rate of cleavage of the scissile bond Arg180-Val181. We conclude that binding to two substrate-binding exosites one on the heavy chain and the other on the light chain of FXIa is required to mediate the formation of the Michaelis complex and efficient cleavages of the two spatially separated scissile bonds of FIX. PMID:17676929

  20. Role of κ→λ light-chain constant-domain switch in the structure and functionality of A17 reactibody

    SciTech Connect

    Ponomarenko, Natalia; Belogurov, Alexey Jr; Fedorova, Olga S.; Dubina, Michael; Golovin, Andrey; Lamzin, Victor; Makarov, Alexander A.; Wilmanns, Matthias

    2014-03-01

    Catalytic antibody variants with κ and λ light-chain constant domains show differences in their crystal structures which lead to subtle changes in catalytic efficiency and thermodynamic parameters as well as in their affinity for peptide substrates. The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophosphate-metabolizing reactibody A17 has been re-engineered by replacing its constant κ light chain by the λ chain (A17λ), and the X-ray structure of A17λ has been determined at 1.95 Å resolution. It was found that compared with A17κ the active centre of A17λ is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the V{sub L} and V{sub H} domains. These V{sub L}/V{sub H} domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126° compared with 138° in A17κ. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17κ and A17λ variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions.

  1. Reactive Carbonyl Species Derived from Omega-3 and Omega-6 Fatty Acids.

    PubMed

    Wang, Yu; Cui, Ping

    2015-07-22

    Inflammation-related reactive oxygen species (ROS) and reactive nitrogen species (RNS) are associated with the development of cancer. ROS and RNS can directly damage biomacromolecules such as proteins, DNA, and lipids. Lipid peroxidation, however, can result in reactive carbonyl species (RCS) that can also modify proteins and DNA. In contrast to an extensive literature on the modification of proteins and DNA from omega-6 fatty acids, there are few studies on RCS generation from other fatty acids, particularly omega-3 fatty acids, which are frequently consumed from the diet and diet supplements. Therefore, a comparison between omega-3 and omega-6 fatty acids has been conducted. LC-MS/MS analysis of carbonyl-dinitrophenylhydrazine (DNPH) standards yielded characteristic fragment ions. Autoxidation products of α-linolenic acid and linoleic acid were then derivatized with DNPH and analyzed by LC-MS/MS. The results showed that α-linolenic acid, an omega-3 fatty acid, generated more acrolein and crotonaldehyde than did linoleic acid, an omega-6 fatty acid. Omega-3 fatty acids might be easily degraded to smaller monoaldehydes or dicarbonyls. Omega-3 fatty acids have been considered as health improvement components for a long time. However, on the basis of the results presented here, use of omega-3 fatty acids should be re-evaluated in vivo for safety purposes.

  2. Elementary Education: What Is Project Omega?

    ERIC Educational Resources Information Center

    Kirman, Joseph M.

    1980-01-01

    Describes Project Omega for Research in Remote Sensing Education, a program in space education designed for inservice training of elementary social studies teachers. Explains a few research areas dealing with the classroom applications of Landsat imagery. (CS)

  3. OMEGA for the Future of Biofuels

    NASA Technical Reports Server (NTRS)

    Trent, Jonathan

    2010-01-01

    OMEGA: Offshore Membrane Enclosure for Growing Algae. To develop a photobioreactor (PBR) for growing algae (Oil, food, fertilizer) that does not compete with agriculture for land (deployed offshore), water or fertilizer (uses/treats wastewater).

  4. Omega-3 Fatty Acids during Pregnancy

    MedlinePlus

    ... omega-3s. They are mostly found in fatty fish like salmon, sardines, and trout. Some eggs are ... and yogurt. Should I Worry About Eating Certain Fish? Because of mercury contamination of our oceans, rivers, ...

  5. Omega-3 Fatty Acid Supplementation During Pregnancy

    PubMed Central

    Greenberg, James A; Bell, Stacey J; Ausdal, Wendy Van

    2008-01-01

    Omega-3 fatty acids are essential and can only be obtained from the diet. The requirements during pregnancy have not been established, but likely exceed that of a nonpregnant state. Omega-3 fatty acids are critical for fetal neurodevelopment and may be important for the timing of gestation and birth weight as well. Most pregnant women likely do not get enough omega-3 fatty acids because the major dietary source, seafood, is restricted to 2 servings a week. For pregnant women to obtain adequate omega-3 fatty acids, a variety of sources should be consumed: vegetable oils, 2 low-mercury fish servings a week, and supplements (fish oil or algae-based docosahexaenoic acid). PMID:19173020

  6. Zooming in on Omega Centauri Stellar Motion

    NASA Video Gallery

    This movie sequence begins with a ground-based image of the giant globular star cluster Omega Centauri and zooms very tightly in to a Hubble Space Telescope image of the central region of the clust...

  7. Optical Omega network: a compact implementation technique

    NASA Astrophysics Data System (ADS)

    Wong, K. W.; Cheng, L. M.

    1995-10-01

    We propose a technique for the compact implementation of an optical Omega network. This technique utilizes the concept that both the perfect-shuffle interconnection and the switching stages can be realized by the same procedures, i.e., duplicate, shift, superimpose, and mask. As a result, a single set of optics is sufficient to realize the whole Omega network in a time-multiplexed recursive manner. Optical setups were designed and a proof-of-principle experiment was performed.

  8. Omega-3 fatty acids and cardiovascular disease.

    PubMed

    Jain, A P; Aggarwal, K K; Zhang, P-Y

    2015-01-01

    Cardioceuticals are nutritional supplements that contain all the essential nutrients including vitamins, minerals, omega-3-fatty acids and other antioxidants like a-lipoic acid and coenzyme Q10 in the right proportion that provide all round protection to the heart by reducing the most common risks associated with the cardiovascular disease including high low-density lipoprotein cholesterol and triglyceride levels and factors that contribute to coagulation of blood. Omega-3 fatty acids have been shown to significantly reduce the risk for sudden death caused by cardiac arrhythmias and all-cause mortality in patients with known coronary heart disease. Omega-3 fatty acids are also used to treat hyperlipidemia and hypertension. There are no significant drug interactions with omega-3 fatty acids. The American Heart Association recommends consumption of two servings of fish per week for persons with no history of coronary heart disease and at least one serving of fish daily for those with known coronary heart disease. Approximately 1 g/day of eicosapentaenoic acid plus docosahexaenoic acid is recommended for cardio protection. Higher dosages of omega-3 fatty acids are required to reduce elevated triglyceride levels (2-4 g/day). Modest decreases in blood pressure occur with significantly higher dosages of omega-3 fatty acids.

  9. Omega-3 fatty acids and cardiovascular disease.

    PubMed

    Jain, A P; Aggarwal, K K; Zhang, P-Y

    2015-01-01

    Cardioceuticals are nutritional supplements that contain all the essential nutrients including vitamins, minerals, omega-3-fatty acids and other antioxidants like a-lipoic acid and coenzyme Q10 in the right proportion that provide all round protection to the heart by reducing the most common risks associated with the cardiovascular disease including high low-density lipoprotein cholesterol and triglyceride levels and factors that contribute to coagulation of blood. Omega-3 fatty acids have been shown to significantly reduce the risk for sudden death caused by cardiac arrhythmias and all-cause mortality in patients with known coronary heart disease. Omega-3 fatty acids are also used to treat hyperlipidemia and hypertension. There are no significant drug interactions with omega-3 fatty acids. The American Heart Association recommends consumption of two servings of fish per week for persons with no history of coronary heart disease and at least one serving of fish daily for those with known coronary heart disease. Approximately 1 g/day of eicosapentaenoic acid plus docosahexaenoic acid is recommended for cardio protection. Higher dosages of omega-3 fatty acids are required to reduce elevated triglyceride levels (2-4 g/day). Modest decreases in blood pressure occur with significantly higher dosages of omega-3 fatty acids. PMID:25720716

  10. Omega-3-carboxylic acid (Epanova) for hypertriglyceridemia.

    PubMed

    Zhao, Alan; Lam, Sum

    2015-01-01

    Hypertriglyceridemia is a prevalent yet under-addressed condition, often seen in association with uncontrolled diabetes mellitus, obesity, and physical inactivity. The control of triglyceride (TG) levels is essential to prevent the development of coronary artery disease and pancreatitis associated with hypertriglyceridemia. Omega-3-carboxylic acid (Epanova) is the third prescription omega-3 fatty acid product approved in the United States as an adjunct to diet for treating severe hypertriglyceridemia (≥ 500 mg/dL). At the approved dosage, it reduced baseline serum TG levels by 25-30% in a placebo-controlled study. It reduced serum TG levels by an additional 8-15% in patients who were already taking statin therapy. It appeared to have a better bioavailability profile compared with an equivalent dose of omega-3-acid ethyl ester (Lovaza) in both low-fat and high-fat diets. However, evidence behind the effects of omega-3-carboxylic acid on cardiovascular morbidity and mortality, and pancreatitis risk, is lacking. Overall, it is well tolerated, but may induce common gastrointestinal side effects, such as abdominal pain, nausea, and diarrhea. At this time, omega-3-carboxylic acid is an alternative adjunct therapy (in addition to diet) for hypertriglyceridemia. Its potential clinical benefits over other omega-3 formulations have yet to be evaluated.

  11. Topoisomerase I deficiency causes RNA polymerase II accumulation and increases AID abundance in immunoglobulin variable genes.

    PubMed

    Maul, Robert W; Saribasak, Huseyin; Cao, Zheng; Gearhart, Patricia J

    2015-06-01

    Activation-induced deaminase (AID) is a DNA cytosine deaminase that diversifies immunoglobulin genes in B cells. Recent work has shown that RNA polymerase II (Pol II) accumulation correlates with AID recruitment. However, a direct link between Pol II and AID abundance has not been tested. We used the DT40 B-cell line to manipulate levels of Pol II by decreasing topoisomerase I (Top1), which relaxes DNA supercoiling in front of the transcription complex. Top1 was decreased by stable transfection of a short hairpin RNA against Top1, which produced an accumulation of Pol II in transcribed genes, compared to cells transfected with sh-control RNA. The increased Pol II density enhanced AID recruitment to variable genes in the λ light chain locus, and resulted in higher levels of somatic hypermutation and gene conversion. It has been proposed by another lab that AID itself might directly suppress Top1 to increase somatic hypermutation. However, we found that in both AID(+/+) and AID(-/-) B cells from DT40 and mice, Top1 protein levels were identical, indicating that the presence or absence of AID did not decrease Top1 expression. Rather, our results suggest that the mechanism for increased diversity when Top1 is reduced is that Pol II accumulates and recruits AID to variable genes.

  12. Genomic organization and expression of immunoglobulin genes in the Chinese hamster (Cricetulus griseus).

    PubMed

    Qin, T; Zhu, H; Wang, D; Hao, H; Du, W

    2015-01-01

    In science, the hamsters are widely used as a model for studying the human diseases because they display many features like humans. The utility of the Chinese hamster as a biology model can be further enhanced by further characterization of the genes encoding components of the immune system. Here, we report the genomic organization and expression of the Chinese hamster immunoglobulin heavy and light chain genes. The Chinese hamster IgH locus contains 268 VH segments (132 potentially functional genes, 12 ORFs and 124 pseudogenes), 4 DH segments, 6 JH segments, four constant region genes (μ, γ, ε and α) and one reverse δ remnant fragment. The Igκ locus contains only a single Cκ gene, 4 Jκ segments and 48 Vκ segments (15 potentially functional genes and 33 pseudogenes), whereas the Igλ locus contains 4 Cλ genes, but only Cλ 3 and Cλ 4 each preceded by a Jλ gene segment. A total of 49 Vλ segments (39 potentially functional genes, 3 ORFs and 7 pseudogenes) were identified. Analysis of junctions of the recombined V(D)J transcripts reveals complex diversity in both expressed H and κ sequences, but the microhomology-directed VJ recombination obviously results in very limited diversity in the Chinese hamster λ gene despite more potential germline-encoded combinatorial diversity. This is the first study to make a comprehensive analysis of the Ig genes in the Chinese hamster, which provides insights into the Ig genes in placental mammals.

  13. A comparative overview of immunoglobulin genes and the generation of their diversity in tetrapods.

    PubMed

    Sun, Yi; Wei, Zhiguo; Li, Ning; Zhao, Yaofeng

    2013-01-01

    In the past several decades, immunoglobulin (Ig) genes have been extensively characterized in many tetrapod species. This review focuses on the expressed Ig isotypes and the diversity of Ig genes in mammals, birds, reptiles, and amphibians. With regard to heavy chains, five Ig isotypes - IgM, IgD, IgG, IgA, and IgE - have been reported in mammals. Among these isotypes, IgM, IgD, and IgA (or its analog, IgX) are also found in non-mammalian tetrapods. Birds, reptiles, and amphibians express IgY, which is considered the precursor of IgG and IgE. Some species have developed unique isotypes of Ig, such as IgO in the platypus, IgF in Xenopus, and IgY (ΔFc) in ducks and turtles. The κ and λ light chains are both utilized in tetrapods, but the usage frequencies of κ and λ chains differ greatly among species. The diversity of Ig genes depends on several factors, including the germline repertoire and recombinatorial and post-recombinatorial diversity, and different species have evolved distinct mechanisms to generate antibody diversity.

  14. Assessment by Southern blot analysis of UV-induced damage and repair in human immunoglobulin genes.

    PubMed

    Bianchi, M S; Bianchi, N O; de la Chapelle, A

    1990-09-01

    Irradiation of DNA with UV light induces pyrimidine dimers and (6-4) photoproducts. The presence of one of these photolesions in the restriction site of a given endonuclease inhibits DNA cleavage and induces the formation of fragments by incomplete DNA digestion which appear as additional, facultative bands in Southern hybridization autoradiograms. The number and size of these fragments show a positive correlation with the UV dose. The response to UV light of immunoglobulin light-chain constant kappa and heavy-chain constant mu genes was analyzed with 2 specific probes. Constant kappa and mu genes when irradiated as part of the chromatin of living lymphocytes showed a UV sensitivity similar to that of naked DNA. The same genes from granulocytes had 50-60 times lower UV sensitivity. When cells were allowed to repair photolesions for 24 h the facultative bands from granulocytes disappeared indicating that these cells were able to remove photolesions from constant kappa and mu genes. Facultative bands from lymphocytes showed a smaller decrease of density after 24 h repair. This suggests that lymphocytes are less efficient than granulocytes i